Tumor Suppressor Killin

ABSTRACT

The present invention relates to a new tumor suppressor, designated Killin. Also described are diagnostic and therapeutic uses of the Killin protein and the killin gene, alone or in combination with traditional cancer therapies.

The present application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 60/716,691, filed Sep. 13, 2005, the entirecontents of which are hereby incorporated by reference.

The government owns rights in the present invention pursuant to fundingfrom the National Institutes of Health under grant no. RO1 CA105024.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates to the fields of oncology, genetics andmolecular biology. More particular the invention relates to theidentification, on human chromosome 10, of a p53-related tumorsuppressor gene designated as Killin.

II. Related Art

Oncogenesis is a multistep biological process, which is presently knownto occur by the accumulation of genetic damage. On a molecular level,the process of tumorigenesis involves the disruption of both positiveand negative regulatory effectors (Weinberg, 1989). The molecular basisfor human colon carcinomas has been postulated, by Vogelstein andcoworkers (1990), to involve a number of oncogenes, tumor suppressorgenes and repair genes. Similarly, defects leading to the development ofretinoblastoma have been linked to another tumor suppressor gene (Lee etal., 1987). Still other oncogenes and tumor suppressors have beenidentified in a variety of other malignancies. Unfortunately, thereremains an inadequate number of treatable cancers, and the effects ofcancer are catastrophic—over half a million deaths per year in theUnited States alone.

p53 is the most frequently mutated, disrupted, and/or allelically losttumor suppressor gene in human cancer, and it has been a focal point forintensive cancer research (Levine, 1997; Vogelstein et al., 2000;Vousden and Prives, 2005). Functionally, p53 works as a sequencedependent transcription factor, which upon activation by genotoxicstresses such as DNA damages regulates the expression of a set of targetgenes that are involved in cell growth control and apoptosis (El-Deiry,1998; Yu et al., 1999; Vousden and Lu, 2002; Liang and Pardee, 2003). Incontrast to a large number of p53 target genes that were implicated incell apoptosis, activation of cell cycle arrest at G1 by p53 resultspredominantly from the induction of p21 (Deng et al., 1995), whereas p21as well as GADD45 and 14-3-3 proteins were also shown to be involved inG2-M arrest (Taylor and Stark, 2001). Among the known p53 target genesimplicated in apoptosis, a family of Bcl-2 related genes, such as bax,puma and noxa, are the best characterized and thought to work through amitochondria-dependent death pathway (Yu and Zhang, 2005).

Through a genetic approach using somatic gene knockout strategy, it wasshown that cellular choice between growth arrest and death upon p53activation appears to depend on at least two factors. For cell typesthat undergo p53-mediated G1 arrest, elimination of p21 sensitizes cellsto die (Polyak et al., 1996; Yu et al., 2003). In such cases, p21clearly plays a protective role in apoptosis. In cell types that areprone to apoptosis upon p53 activation, transacting death-inducingfactors are dominant over p21-mediated protection (Polyak et al., 1996;Yu et al., 2003). In the case of p21-mediated G1 arrest which protectscells from p53 induced apoptosis, one possible explanation could be thatthe apoptosis initiating event(s) require cells to enter S-phase.Supporting evidence for such S-phase-coupled apoptosis include findingsthat forced S-phase entry by unrestricted E2F activity can trigger theactivation of caspases and apoptosis (Nahle et al., 2002; Gottifredi andPrives, 2005). Conceivably, DNA damage can happen to cells at any phaseduring the cell cycle. The induction of either p21 in cells at G1, orp21, GADD45 and 14-3-3 at G2/M phase by p53 will lead to growth arrestat the respective cell cycle phases. However, little is known aboutp53-mediated checkpoint control during S-phase where cells would run thehighest risk of incorporating mutations after sustained DNA damage. Itis logical that apoptosis would be the best choice for eliminating thesecells. summary of the invention

SUMMARY OF THE INVENTION

Thus, in accordance with the present invention, there is provided anisolated polynucleotide encoding a polypeptide having an amino acidsequence of SEQ ID NO:1 The polynucleotide may further have the nucleicacid sequence of SEQ ID NO:2 or a complement thereof. In addition, thepolynucleotide may further comprise a promoter operable in eukaryoticcells. The promoter may be heterologous to the coding sequence, and maybe selected from the group consisting of hsp68, SV40, CMV IE, MKC,GAL4_(UAS), HSV and β-actin. The promoter may be a tissue specificpromoter or inducible promoter.

Also provided is a nucleic acid of about 15 to about 5000 base pairscomprising from about 15 contiguous base pairs of SEQ ID NO:2, or thecomplement thereof. The nucleic acid may comprise about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250,500, 1000, 2500, or 4000 contiguous base pairs of SEQ ID NO:2, or thecomplement thereof. The nucleic acid itself may be 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95 100, 150, 200, 250, 300, 400, 500, 1000, 2000,3000, 4000, or 5000 base pairs.

In another embodiment, there is provided a peptide comprising about10-50 contiguous amino acids of SEQ ID NO:1, or more specifically, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 contiguousamino acids of SEQ ID NO:1. The peptide may comprise residues 8-49 of ofSEQ ID NO:1, or the full length sequence of SEQ ID NO:1.

In yet another embodiment, there is provided an expression cassettecomprising a polynucleotide encoding a polypeptide having the sequenceof SEQ ID NO:1 or a fragment thereof, wherein the polynucleotide isunder the control of a promoter operable in eukaryotic cells. Thepromoter may be heterologous to the coding sequence, and may be selectedfrom the group consisting of hsp68, SV40, CMV IE, MKC, GAL4_(UAS), HSVand β-actin. The promoter may be a tissue specific promoter or induciblepromoter. The expression cassette may be contained in a viral vector,for example, a retroviral vector, an adenoviral vector, andadeno-associated viral vector, a vaccinia viral vector, and aherpesviral vector. The expression cassette may further comprise apolyadenylation signal, may further comprise a second polynucleotideencoding a second polypeptide which may be under the control of a secondpromoter.

In still yet another embodiment, there is provided a method forsuppressing growth of a cancer cell comprising contacting the cells withan expression cassette comprising a polynucleotide encoding apolypeptide having the sequence of SEQ ID NO:1 or a fragment thereof,wherein the polynucleotide is under the control of a promoter operablein eukaryotic cells. The promoter may be heterologous to thepolynucleotide sequence, such as hsp68, SV40,

CMV, MKC, GAL4_(UAS)HSV and β-actin. The may be tissue specific promoteror an inducible promoter. The expression cassette may be contained in aviral vector, for example, a retroviral vector, an adenoviral vector,and adeno-associated viral vector, a vaccinia viral vector, and aherpesviral vector. The expression cassette may further comprise apolyadenylation signal, a second polynucleotide encoding a secondpolypeptide which may be under the control of a second promoter.

In an additional embodiment, there is provided a cell comprising anexpression cassette comprising a polynucleotide encoding a polypeptidehaving the sequence of SEQ ID NO:1 or a fragment thereof, wherein thepolynucleotide is under the control of a promoter operable in eukaryoticcells.

In still an additional embodiment, there is provided a monoclonalantibody that binds immunologically to a polypeptide having the sequenceof SEQ ID NO:1, or an immunologic fragment thereof. The antibody mayfurther comprise a detectable label, for example, a fluorescent label, achemiluminescent label, a radiolabel and an enzyme.

In another embodiment, there is provided a hybridoma cell that producesa monoclonal antibody that binds immunologically to a polypeptide havingthe sequence of SEQ ID NO:1, or an immunologic fragment thereof. Alsoprovided is a polyclonal antisera, antibodies of which bindimmunologically to a polypeptide having the sequence of SEQ ID NO:1, oran immunologic fragment thereof.

In still another embodiment, there is provided a method of diagnosing acancer comprising the steps of (i) obtaining a tissue sample from asubject; and (ii) assessing the expression or structure of Killin incells of the sample. The cancer may be selected from the groupconsisting of brain, lung, liver, spleen, kidney, lymph node, smallintestine, pancreas, blood cells, colon, stomach, breast, endometrium,prostate, testicle, ovary, skin, head and neck, esophagus, bone marrowand blood cancer. The method may comprise assessing Killin expression orKillin structure. The sample may be a tissue or fluid sample. Assessingmay comprise assaying for a Killin-encoding nucleic acid from thesample, such as by subjecting the sample to conditions suitable toamplify the nucleic acid. Assessing may comprise contacting the samplewith an antibody that binds immunologically to a Killin polypeptide,such as by ELISA. Assessing may comprise evaluating the level of Killinexpression, for example, by comparing the expression of Killin with theexpression of Killin in non-cancer samples. Assessing may compriseevaluating the structure of the Killin gene or transcript, for example,by sequencing, wild-type oligonucleotide hybridization, mutantoligonucleotide hybridization, SSCP, PCR and RNase protection, includinguse of an array on a chip or wafer.

In still another embodiment, there is provided a method for altering thephenotype of a tumor cell comprising the step of administering to a cella tumor suppressor designated Killin or a fragment thereof underconditions permitting the uptake of the tumor suppressor by the tumorcell. The tumor cell may be derived from a tissue selected from thegroup consisting of brain, lung, liver, spleen, kidney, lymph node,small intestine, blood cells, pancreas, colon, stomach, breast,endometrium, prostate, testicle, ovary, skin, head and neck, esophagus,bone marrow and blood tissue. The phenotype may be selected from thegroup consisting of apoptosis, angiogenesis, proliferation, migration,contact inhibition, soft agar growth and cell cycling. The tumorsuppressor may be is encapsulated in a liposome.

A further embodiment comprises a method for altering the phenotype of atumor cell comprising the step of contacting the cell with a nucleicacid (i) encoding a tumor suppressor designated Killin or a fragmentthereof and (ii) a promoter active in the tumor cell, wherein thepromoter is operably linked to the region encoding the tumor suppressor,under conditions permitting the uptake of the nucleic acid by the tumorcell. The tumor cell may be derived from a tissue selected from thegroup consisting of brain, lung, liver, spleen, kidney, lymph node,small intestine, blood cells, pancreas, colon, stomach, breast,endometrium, prostate, testicle, ovary, skin, head and neck, esophagus,bone marrow and blood tissue. The phenotype may be selected from thegroup consisting of apoptosis, angiogenesis, proliferation, migration,contact inhibition, soft agar growth or cell cycling. The nucleic acidmay be encapsulated in a liposome. The nucleic acid may comprise a viralvector selected from the group consisting of retrovirus, adenovirus,adeno-associated virus, vaccinia virus and herpesvirus. The nucleic acidmay be encapsulated in a viral particle.

Yet another embodiment provides a method for treating subject withcancer comprising the step of administering to the subject a tumorsuppressor designated Killin or a fragment thereof. The tumor cell maybe derived from a tissue selected from the group consisting of brain,lung, liver, spleen, kidney, lymph node, small intestine, blood cells,pancreas, colon, stomach, breast, endometrium, prostate, testicle,ovary, skin, head and neck, esophagus, bone marrow and blood tissue. Thesubject may be a human. The method may further comprise treating thesubject with a second anti-cancer therapy, such as radiation therapy,gene therapy, hormonal therapy, immunotherapy, toxin therapy or surgery.The gene therapy may be p53 gene therapy.

In still another embodiment, there is provided a method for treating asubject with cancer comprising the step of administering to the subjecta nucleic acid (i) encoding a tumor suppressor designated Killin or afragment thereof and (ii) a promoter active in eukaryotic cells, whereinthe promoter is operably linked to the region encoding the tumorsuppressor. The tumor cell may be derived from a tissue selected fromthe group consisting of brain, lung, liver, spleen, kidney, lymph node,small intestine, blood cells, pancreas, colon, stomach, breast,endometrium, prostate, testicle, ovary, skin, head and neck, esophagus,bone marrow and blood tissue. The subject may be a human. The method mayfurther comprise treating the subject with a second anti-cancer therapy,such as radiation therapy, gene therapy; hormonal therapy,immunotherapy, toxin therapy or surgery. The gene therapy may be p53gene therapy.

In a further embodiment, there is provided a non-human transgeniceukaryote lacking a functional Killin gene, for example, wherein theeukaryote is a mammal. Another embodiment is a non-human transgeniceukaryote that overexpresses Killin as compared to a similarnon-transgenic eukaryote, for example, wherein the eukaryote is amammal.

A method of screening a candidate substance for anti-tumor activitycomprising the steps of (i) providing a cell lacking functional Killinpolypeptide; (ii) contacting the cell with the candidate substance; and(iii) determining the effect of the candidate substance on the cell. Thecell may be a tumor cell, such as a tumor cell having a mutation in thecoding region of Killin. The mutation may be a deletion mutant, aninsertion mutant, a frameshift mutant, a nonsense mutant, a missensemutant or splice mutant. Determining may comprise comparing one or morecharacteristics of the cell in the presence of the candidate substancewith characteristics of a cell in the absence of the candidatesubstance. The characteristic may be selected from the group consistingof proliferation, metastasis, apoptosis, contact inhibition, soft agargrowth, cell cycle regulation, tumor formation, tumor progression andtissue invasion. The candidate substance may be a chemotherapeutic orradiotherapeutic agent, or selected from a small molecule library. Themay be contacted in vitro or in vivo.

In still yet another embodiment, there is provided an isolated andpurified nucleic acid that hybridizes, under high stringency conditions,to a DNA segment comprising SEQ ID NO:2.

Another embodiment comprises a method of screening a candidate substancefor anti-tumor activity comprising the steps of (i) providing a cellexpression a functional Killin peptide or polypeptide; (ii) contactingthe cell with the candidate substance; and (iii) determining Killin DNAbinding or nuclear localizaion, wherein an increase in Killin DNAbinding or nuclear localization, as compared to a similar cell nottreated with the candidate substance, indicates that the candidatesubstance has anti-tumor activity.

In yet an additional embodiment, there is provided a nucleic acidsegment comprising SEQ ID NO:3. Also provided is a method of screeningfor an activator of Killin expression comprising (i) providing a cellcomprising a Killin promoter operably linked to a nucleic acid segmentencoding expressable marker; (ii) contacting said cell with a candidatesubstance; and (iii) assessing the expression of said marker, wherein anincrease in expression of said marker, as compared to expression in acell not contacted with said candidate substance, identifies saidcandidate substance as an activator of Killin expression. The cell maybe a eukaryotic cell. The candidate substance may be a protein, apeptide, an organopharmaceutical, a lipid, a carbohydrate or a nucleicacid. The expressable marker may be an enzyme or a fluorescent orchemilluminescent protein.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

These, and other, embodiments of the invention will be betterappreciated and understood when considered in conjunction with thefollowing description and the accompanying drawings. It should beunderstood, however, that the following description, while indicatingvarious embodiments of the invention and numerous specific detailsthereof, is given by way of illustration and not of limitation. Manysubstitutions, modifications, additions and/or rearrangements may bemade within the scope of the invention without departing from the spiritthereof, and the invention includes all such substitutions,modifications, additions and/or rearrangements.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein:

FIGS. 1A-D—Identification and Confirmation of killin (G101) as a Novelp53 Target Gene. (FIG. 1A) Identification of killin by FDD. p53-3, aH1299 lung cancer cell line with a Tet-repressible p53 expressionconstruct was either uninduced (+tet) or induced (−tet) for wild-typep53 for the time points indicated. p53 dependent expression of killin(G101) was detected by FDD with a G-anchored primer and arbitrary primerHAP-101 (indicated by arrow). (FIG. 1B) Northern Blot confirmation.killin cDNA was recovered from the FDD gel, cloned and used as a probefor Northern blot confirmation of its induction by p53 which wasverified by western blot analysis. rRNA loading control was shown as thebottom panel. (FIG. 1C) p53 dependence of killin expression. To rule outthe effect of tetracycline, parental H1299 cell line (p53 null) wasgrown also in the absence of tet for 24 hours and compared for killinexpression by Northern blot analysis with p53-3 cells (H1299 withinducible wt p53) either without (+tet) and with (−tet) p53 inductionfor the same duration. The induction of p53 protein was confirmed bywestern blot analysis. rRNA loading control was shown as the bottompanel. (FIG. 1D) Predicted amino acid sequence of Killin with putativenuclear localization sequence (NLS) underlined.

FIGS. 2A-B—Killin is Localized in Close Proximity to pTENTumor-Suppressor Gene and is Transcriptionally Activated by p53. (FIG.2A) Chromosomal locus of killin. The 194 by intergenic region separatingkillin and pTEN contains a divergent promoter with a p53 consensusbinding site (underlined). Note killin is encoded by a single exon of4.1 kb, whereas pTEN is encoded by multiple exons and introns spanningover 100 kb. (FIG. 2B) Lucifersase reporter assays showing that the 140by killin promoter sequence containing the conserved p53 binding site(pGL3-Killin) conferred a dramatic p53-dependent transcriptionactivation, whereas mutations at the key p53 consensus bases within theKillin promoter (pGL3-Killin-mutant) greatly decreased the p53 effect.Co-transfected vectors expressing either wild-type (pCep4-p53wt) or aDNA binding mutant of p53 (R248W) (pCep4-p53mut), as well as the vectorcontrol (pCep4) were as indicated. FIGS. 3A-C—Killin is a NuclearProtein. (FIG. 3A) DLD-1 cell lines stably expressing either inducibleGFP or GFP-Killin was visualized by fluorescence microscopy after 16 hrsof induction (−tet). Note that GFP-Killin is localized exclusively inthe nucleus, whereas GFP is expressed throughout the cells. (FIG. 3B)pEGFP-C1 vector expressing an in-frame GFP-Killin fusion protein wastransiently transfected into Cos-1 cells. One day (left panels) andthree days (right panels) after transfection, cells expressing theGFP-Killin were visualized under a Zeiss fluorescent microscope (20×).Compared to nuclear staining with DAPI, GFP-Killin was clearly localizedin the nucleus with focal distribution pattern (upper left). Inapoptotic cells (upper right), GFP-Killin appeared to be associated withcondensed apoptotic chromotin. (FIG. 3C) Confocal fluorescentmicroscopy. Cos-1 cells after transiently transfected with eitherGFP-PCNA or GFP-Killin for 16 hrs showed distribution pattern as nuclearfoci.

FIGS. 4A-B—Killin is Necessary for p53-mediated Apoptosis. (FIG. 4A)RNAi knockdown of killin expression blocks p53 induced Caspase-3activation and PARP cleavage. p53-3 cells stably transfected with eitherpSuper-RNAi vector alone or pSuper-RNAi-killin were either non-induced(+tet) or induced (−tet) for 24 hrs. The induction of p53 and p21 wereconfirmed by Western blot analysis with β-actin as a control for equalsample loading. RNAi knockdown of killin expression in p53-3 cells ledto not only diminished p53-dependent expression of killin mRNA asdetermined by real time RT-PCR, but also inhibition of Caspase-3activation and cleavage of PARP analyzed by Western blot. RNAi knockdownof killin expression had little effect on p53 induction and its effecton p21 expression. (FIG. 4B) RNAi knockdown of killin expression blocksp53-mediated Apoptosis. FACS analysis of p53-3 cells stably transfectedwith either pSuper-RNAi-killin or pSuper vector control confirmed thatblocking p53-dependent killin expression would essentially prevent cellsfrom apoptosis, without affecting G1 arrest mediated by p21. The p53induction time following tetracycline withdraw was as indicated. Theresults were representative of multiple clones of cells in duplicatedexperiments.

FIGS. 5A-C—Killin is Sufficient for Cell Apoptosis. (FIG. 5A) Killinexpression causes rapid inhibition in cell proliferation. Cellproliferation rate for DLD-1 cells with tetracycline regulatedexpression of either GFP-Killin or GFP alone were compared with (−tet)or without induction (+tet). Both attached (live) and detached (dying)cells were counted and combined for each time point as indicated. (FIG.5B) Killin expression leads to rapid cell cycle arrest followed bymassive cell apoptosis. FACS analysis of DLD-1 cells following inducibleexpression of GFP-Killin showed little decrease in S-phase DNA contentor increase in G1 or G2/M DNA content during the first 48 firs ofGFP-Killin induction, when growth arrest became apparent (FIG. 5A).Massive apoptosis based on sub-G1 DNA content was apparent after 72 hrspost induction of GFP-Killin. (FIG. 5C) The induction of GFP andGFP-Killin were also visualized by fluorescence microscopy, whereascells were depicted by phase-contrast. Note massive cell death(reflectory detached cells) induced by GFP-Killin (−tet) after 72 hrs,compared to either non-induced cells (+tet) or GFP alone induced cellsduring the same time period.

FIGS. 6A-D—Killin is a High Affinity DNA Binding Protein. (FIG. 6A) Thefull-length native Killin is a DNA binding protein. In vitro transcribedand translated Killin (K) or vector alone (V) were labeled with ³⁵S andincubated with either single-stranded (ss) or double-stranded (ds) DNAcellulose. After washing with PBS, bound proteins were resolved on a 15%SDS-PAGE gel and visualized by autoradiography. Killin (20 Kda), but notthe non-specific protein (100 Kda) from the vector alone wasspecifically retained by DNA cellulose. (FIG. 6B) Bacterial geneticscreen and serial deletion analysis of the functional domain of Killin.pQE32 bacterial expression vectors encoding either the full-lengthN-terminal His-tagged Killin (1-178 aa), or truncated Killin asindicated were transformed into either XL-1 Blue (lac I^(q) withrepression) or GH1 (wild-type lac I without repression) competent cellsand selected with ampicillin in the absence of IPTG. While all plasmidsgave numerous colonies when transformed under repressed condition inXL-1 blue, Killin deletions that retained the ability to kill E. coliwere scored for their ability to inhibit colony formation in GH1 cells.Similar deletion mutants of Killin were made into GFP fusion protein inpEGFP-C 1 expression vector and transiently transfected into H1299 cellsto score for their ability to induced cell apoptosis based on florescentmicroscopy of the nuclear condensation. (FIG. 6C) Amino acid sequence ofKillin/N8-50 peptide with the minimum 8-49 aa residues underlined. Thechemically synthesized Killin/N8-50 was analyzed on a 15% SDS PAGE byCoomassie Blue staining with the amount of peptide as indicated. (FIG.6D) In vitro DNA binding kinetics of Killin/N8-50 peptide. ³²⁻P endlabeled double-stranded, single-stranded and artificial replication forkDNA templates of 32-35 bases or by in length were each incubated withincreasing concentration of Killin/N8-50 peptide as indicated. Thereactions were resolved on a 6% TBE PAGE gel. The Killin-DNA bindingkinetics was quantified by counting the radioactivity of the complexformed (upper retained band) from each reaction in duplicate.

FIGS. 7A-B—Stability of Killin/N8-50-DNA Complex and Inhibition of invitro DNA Replication by Killin/N8-50 Peptide. (FIG. 7A) Killin/N8-50and DNA forms a non-covalently linked stable complex. Equal amount (500ng) of either double-stranded (replication form) or single-stranded(viral form) PhiX174 bacterial phage DNA was incubated with either 1 μgof BSA or Killin/N8-50 peptide. The stability of Killin-DNA complex, inthe presence of either 6 M urea, 150 mM EDTA or 0.1% SDS as indicated,was then assessed on a 0.8% TAE agarose gel after ethidium bromidestaining. (FIG. 7B) Inhibition of eukaryotic DNA replication in vitro byKillin/N8-50 peptide. The concentration of Killin/N8-50 peptide used wasfrom 1, 1.6, 2.4, 3.2, 4.8, 6.4, 8 to 16 μM, as indicated.

FIG. 8—Representative FDD (in gray scale) detecting p53 regulatedcahnges in gene expression following tetracycline removal from DLD-1 orH1299 cells with inducible wild-type p53. Four RNA samples representing9 and 12 hours with Tet (left two lanes) and without Tet (right twolanes), respectively were compared. Primer combinations detecting thechanges in gene expression are as indicated (e.g., G3 =G-anchor +HAP-3,etc.). Four of the clearly induced genes (G20, G54, G63 and G116) aftersequencing turned out to be p53 itself, validating the comprehensivenessand precision of the inventors' FDD platform. The nature of other p53induced genes is summarized in Table 6.

FIG. 9—Positive Feedback regulation of p53 by Killin. Western blotanalysis of DLD-1 cells with an inducible GFP-Killin led to increase inp53 as well as its major target gene products, such as p21 and HDM-2proteins. Cells were either non-induced (+tet) or induced (−tet) for theduration as indicated. Actin was used as a control for equal sampleloading. GFP-Killin was detect with an antibody to GFP.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. The PresentInvention

The inventors have identified of a novel p53 target gene, killin, whichencodes a small nuclear DNA binding protein with a high affinity to bothdouble-stranded and single-stranded DNA. Killin is not only necessary,but sufficient for mediating p53-induced growth arrest and apoptosis.Genetic and biochemical analysis reveal that the DNA binding domain ofKillin resides within 42 amino acid residues near the N-terminus of theprotein which can inhibit DNA synthesis in vitro and S-phase arrestcoupled to apoptosis in vivo. Thus, Killin represents the first p53target gene that is directly involved in S-phase checkpointcontrol-coupled apoptosis. These findings also help to explain theapparent paradox of p21 being both a growth and death inhibitor, sinceG1 arrest triggered by p21 can prevent cells from S-phase entry, therebyescaping the fate of death through S-phase checkpoint control mediatedby Killin.

Compelling evidence from cell biological, genetic and biochemicalanalysis of the gene suggests the following possible mechanism of actionfor Killin in mediating tumor-suppressor p53 functions. Upon inductionby p53 during S-phase, Killin functions in the cell nucleus as a DNAsynthesis inhibitor via its high affinity to both double-stranded andsingle-stranded DNA (e.g., at the replication forks) and thereby causesS-phase arrest, which in turn triggers subsequent cell apoptosis. Thus,Killin-mediated checkpoint control at S-phase would complement those atG1 mediated by p21, and G2-M phase by p21, GADD45 and 14-3-3, andprovides a fool-proof mechanism for p53 in preventing precancerous cellsfrom replicating their DNA content. Thus, Killin represents the firstp53 target gene that is directly and functionally involved in S-phasecheckpoint control which seems to be coupled to apoptosis, in contrastto p21 mediated G1 arrest, which is anti-apoptotic. The unique functionof Killin in coupling S-phase arrest with apoptosis may also explain whyp21-mediated G1 arrest can be anti-apoptotic. Conceivably, prevention ofcells from S-phase entry by p21 would spare cells from Killin-mediatedinhibition of DNA synthesis. Without stalled replication forks caused byKillin, apoptosis may be avoided. The high affinity of Killin to bothdouble-stranded and single-stranded DNA could also reconcile with thediffusive focal distribution pattern of Killin in S-phase nuclei whichundergo further condensation characteristic of apoptotic cells.

While Killin is not only necessary but sufficient in triggering rapidcell growth arrest, commencement of cell apoptosis induced by Killin(after 48 hours) appeared to be rather delayed, as compared to theeffect of p53 induction (within 48 hours). The delayed onset of cellapoptosis seems to suggest that Killin-mediated growth arrest at S-phasemay lead to subsequent activation of other downstream genes, which maycooperate in triggering a full apoptotic response. In contrast, suchgenes may be coordinately induced by p53 as immediate targets along withkillin. This conjecture appeared to be supported by an intriguing pieceof evidence of ours which showed that Killin expression alone was ableto induce not only several major p53 target genes that the inventorshave examined, such as hdm2 and p21, but also the endogenous p53 (FIG.9). Thus, Killin may be part of a positive-feedback loop that isdesigned to amplify p53 functions to ensure precancerous cells areeither completely arrested simultaneously at multiple checkpoints, orkilled through apoptosis. The positive feedback activation of endogenousp53 by Killin conceivably could be mediated by ATR or its relatedkinases, which are known to be activated by genotoxic stresses such asstalled DNA replication forks (Abraham, 2001; Falck et al., 2005). Theinduction of p21 and possible other cell cycle regulators at G2/M byGFP-Killin may also explain the static cell cycle profiles within 48hours following GFP-Killin induction. Future studies should help betterdefine this important regulatory circuitry for p53.

Close inspection of the minimal 41 amino acid Killin peptide sequenceessential for DNA binding in vitro and killing of bacteria in vivo, theinventors noted multiple WXXR and KXXW motifs (FIG. 6C). Although,theoretical protein folding prediction could not provide definitivesecondary structure of the Killin/N8-50 peptide, conceivably theseregular motifs would bring R, K and W residues along the same surfacefor DNA binding should the peptide fold into binary alpha helices thatare connected by the single proline residue within the peptide sequence.The binary DNA binding fingers could allow Killin to bind to more thanone DNA template, causing it to tangle up, which may explain whyDNA-Killin/N8-50 peptide complex had a dramatically retarded mobility onthe gel. Conceivably, tryptophan (W) may interact with purine orpyrimidine bases, while basic amino acid residues arginine (R) andlysine (K) may interact with phosphates in the DNA. The extremely tightbinding of Killin to DNA may prevent DNA synthesis machinery from accessto the template, thus leading to inhibition of DNA synthesis and S-phasearrest. Future structural-functional studies by NMR and sited-directedmutagenesis should help verify or refine our prediction. The shortKillin peptide (41-42 aa) and its potent activity in DNA binding,inhibition of DNA synthesis and ability to trigger apoptosis also makeit a good candidate as a peptide drug for cancer treatment.

The extremely close proximity of killin and pten did not escape notice,since it would also make killin a candidate tumor-suppressor gene. ptenwas originally identified as a candidate tumor-suppressor by positionalcloning from chromosome 10q23 region, which is frequently deleted in avariety of human tumors (Li et al., 1997; Steck et al., 1997). WhilepTEN is encoded by multiple exons spanning over 100 kb, killin residesin a single exon of only 4.1 kb. In fact, the extremely short 194 byintergenic region connecting the two genes contains a divergent promoterthat appears to be p53-responsive for both pten (Stambolic et al., 2001)and killin, with the latter shown here to be completely p53-dependent.Since one logical prediction for a major p53 target gene would be thatsuch a gene could be a tumor-suppressor on its own, mutational analysisin cancer and genetic studies in animal models should help furtherdefine the precise role of Killin in tumor suppression.

II. Killin

According to the present invention, there has been identified a tumorsuppressor encoded by a gene in the pten locus, and designated here asKillin. This molecule is capable of suppressing tumor phenotypes invarious cancers. In addition to the entire Killin molecule, the presentinvention also relates to fragments of the polypeptide that may or maynot retain the tumor suppressing (or other) activity. Fragments,including the N-terminus of the molecule may be generated by geneticengineering of translation stop sites within the coding region(discussed below). Alternatively, treatment of the Killin molecule withproteolytic enzymes, known as proteases, can produces a variety ofN-terminal, C-terminal and internal fragments. Examples of fragments mayinclude contiguous residues of the Killin sequence given in SEQ ID NO:1of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85, 90, 95, 100, 125,150, or 178 amino acids in length. These fragments may be purifiedaccording to known methods, such as precipitation (e.g., ammoniumsulfate), HPLC, ion exchange chromatography, affinity chromatography(including immunoaffinity chromatography) or various size separations(sedimentation, gel electrophoresis, gel filtration).

A. Features of the Polypeptide

The gene for Killin encodes a 178 amino acid polypeptide (SEQ ID NO:1).When the present application refers to the function of Killin or“wild-type” activity, it is meant that the molecule in question has theability to inhibit the transformation of a cell from a normallyregulated state of proliferation to a malignant state, i.e., oneassociated with any sort of abnormal growth regulation, or to inhibitthe transformation of a cell from an abnormal state to a highlymalignant state, e.g., to prevent metastasis or invasive tumor growth.Other phenotypes that may be regulated by the normal Killin gene productare angiogenesis, adhesion, migration, cell-to-cell signaling, cellgrowth, cell proliferation, density-dependent growth,anchorage-dependent growth and others. Determination of which moleculespossess this activity may be achieved using assays familiar to those ofskill in the art. For example, transfer of genes encoding Killin, orvariants thereof, into cells that do not have a functional Killinproduct, and hence exhibit impaired growth control, will identify, byvirtue of growth suppression, those molecules having Killin function.

B. Variants of Killin

Amino acid sequence variants of the polypeptide can be substitutional,insertional or deletion variants. Deletion variants lack one or moreresidues of the native protein which are not essential for function orimmunogenic activity, and are exemplified by the variants lacking atransmembrane sequence described above. Another common type of deletionvariant is one lacking secretory signal sequences or signal sequencesdirecting a protein to bind to a particular part of a cell. Insertionalmutants typically involve the addition of material at a non-terminalpoint in the polypeptide. This may include the insertion of animmunoreactive epitope or simply a single residue. Terminal additions,called fusion proteins, are discussed below.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, such as stabilityagainst proteolytic cleavage, without the loss of other functions orproperties. Substitutions of this kind preferably are conservative, thatis, one amino acid is replaced with one of similar shape and charge.Conservative substitutions are well known in the art and include, forexample, the changes of: alanine to serine; arginine to lysine;asparagine to glutamine or histidine; aspartate to glutamate; cysteineto serine; glutamine to asparagine; glutamate to aspartate; glycine toproline; histidine to asparagine or glutamine; isoleucine to leucine orvaline; leucine to valine or isoleucine; lysine to arginine; methionineto leucine or isoleucine; phenylalanine to tyrosine, leucine ormethionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

The following is a discussion based upon changing of the amino acids ofa protein to create an equivalent or improved molecule. For example,certain amino acids may be substituted for other amino acids in aprotein structure without appreciable loss of interactive bindingcapacity with structures such as, for example, antigen-binding regionsof antibodies or binding sites on substrate molecules. Since it is theinteractive capacity and nature of a protein that defines that protein'sbiological functional activity, certain amino acid substitutions can bemade in a protein sequence, and its underlying DNA coding sequence, andnevertheless obtain a protein with like properties. It is thuscontemplated by the inventors that various changes may be made in theDNA sequences of genes without appreciable loss of their biologicalutility or activity, as discussed below. Table 1 shows the codons thatencode particular amino acids.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In making substitutional variants, the hydropathic index of amino acidsmay be considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte & Doolittle, 1982). It is accepted that therelative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte & Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine *−5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan(−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent and immunologically equivalent protein. In such changes, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine andisoleucine.

Another embodiment for the preparation of polypeptides according to theinvention is the use of peptide mimetics. Mimetics arepeptide-containing molecules that mimic elements of protein secondarystructure. See, for example, Johnson et al., (1993). The underlyingrationale behind the use of peptide mimetics is that the peptidebackbone of proteins exists chiefly to orient amino acid side chains insuch a way as to facilitate molecular interactions, such as those ofantibody and antigen. A peptide mimetic is expected to permit molecularinteractions similar to the natural molecule. These principles may beused, in conjunction with the principles outline above, to engineersecond generation molecules having many of the natural properties ofKillin, but with altered and even improved characteristics.

C. Domain Switching

Domain switching involves the generation of chimeric molecules usingdifferent but, in this case, related polypeptides. By comparing theKillin sequence with other tumor suppressors, one can make predictionsas to the functionally significant regions of these molecules. It ispossible, then, to switch related domains of these molecules in aneffort to determine the criticality of these regions to Killin function.These molecules may have additional value in that these “chimeras” canbe distinguished from natural molecules, while possibly providing thesame function.

D. Fusion Proteins

A specialized kind of insertional variant is the fusion protein. Thismolecule generally has all or a substantial portion of the nativemolecule, linked at the N- or C-terminus, to all or a portion of asecond polypeptide. For example, fusions typically employ leadersequences from other species to permit the recombinant expression of aprotein in a heterologous host. Another useful fusion includes theaddition of a immunologically active domain, such as an antibodyepitope, to facilitate purification of the fusion protein. Inclusion ofa cleavage site at or near the fusion junction will facilitate removalof the extraneous polypeptide after purification. Other useful fusionsinclude linking of functional domains, such as active sites fromenzymes, glycosylation domains, cellular targeting signals ortransmembrane regions.

E. Purification of Proteins

It will be desirable to purify Killin or variants thereof. Proteinpurification techniques are well known to those of skill in the art.These techniques involve, at one level, the crude fractionation of thecellular milieu to polypeptide and non-polypeptide fractions. Havingseparated the polypeptide from other proteins, the polypeptide ofinterest may be further purified using chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity). Analytical methods particularly suitedto the preparation of a pure peptide are ion-exchange chromatography,exclusion chromatography; polyacrylamide gel electrophoresis;isoelectric focusing. A particularly efficient method of purifyingpeptides is fast protein liquid chromatography or even HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide. The term “purified protein or peptide” as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the protein or peptide is purified to any degreerelative to its naturally-obtainable state. A purified protein orpeptide therefore also refers to a protein or peptide, free from theenvironment in which it may naturally occur.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related in asimple matter to molecular weight.

Affinity Chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (alter pH, ionic strength, temperature, etc.).

A particular type of affinity chromatography useful in the purificationof carbohydrate containing compounds is lectin affinity chromatography.Lectins are a class of substances that bind to a variety ofpolysaccharides and glycoproteins. Lectins are usually coupled toagarose by cyanogen bromide. Conconavalin A coupled to Sepharose was thefirst material of this sort to be used and has been widely used in theisolation of polysaccharides and glycoproteins other lectins that havebeen include lentil lectin, wheat germ agglutinin which has been usefulin the purification of N-acetyl glucosaminyl residues and Helix pomatialectin. Lectins themselves are purified using affinity chromatographywith carbohydrate ligands. Lactose has been used to purify lectins fromcastor bean and peanuts; maltose has been useful in extracting lectinsfrom lentils and jack bean; N-acetyl-D galactosamine is used forpurifying lectins from soybean; N-acetyl glucosaminyl binds to lectinsfrom wheat germ; D-galactosamine has been used in obtaining lectins fromclams and L-fuctose will bind to lectins from lotus.

The matrix should be a substance that itself does not adsorb moleculesto any significant extent and that has a broad range of chemical,physical and thermal stability. The ligand should be coupled in such away as to not affect its binding properties. The ligand should alsoprovide relatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand. One of the mostcommon forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

F. Synthetic Peptides

The present invention also describes smaller Killin-related peptides foruse in various embodiments of the present invention. Because of theirrelatively small size, the peptides of the invention can also besynthesized in solution or on a solid support in accordance withconventional techniques. Various automatic synthesizers are commerciallyavailable and can be used in accordance with known protocols. See, forexample, Stewart and Young (1984); Tam et al. (1983); Merrifield (1986);and Barany and Merrifield (1979), each incorporated herein by reference.Short peptide sequences, or libraries of overlapping peptides, usuallyfrom about 6 up to about 35 to 50 amino acids, which correspond to theselected regions described herein, can be readily synthesized and thenscreened in screening assays designed to identify reactive peptides.Alternatively, recombinant DNA technology may be employed wherein anucleotide sequence which encodes a peptide of the invention is insertedinto an expression vector, transformed or transfected into anappropriate host cell and cultivated under conditions suitable forexpression.

G. Antigen Compositions

The present invention also provides for the use of Killin proteins orpeptides as antigens for the immunization of animals relating to theproduction of antibodies. It is envisioned that either Killin, orportions thereof, will be coupled, bonded, bound, conjugated orchemically-linked to one or more agents via linkers, polylinkers orderivatized amino acids. This may be performed such that a bispecific ormultivalent composition or vaccine is produced. It is further envisionedthat the methods used in the preparation of these compositions will befamiliar to those of skill in the art and should be suitable foradministration to animals, i.e., pharmaceutically acceptable. Preferredagents are the carriers are keyhole limpet hemocyannin (KLH) or bovineserum albumin (BSA).

III. Nucleic Acids

The present invention also provides, in another embodiment, genesencoding Killin. A gene for the human Killin molecule has beenidentified. The present invention is not limited in scope to this gene,however, as one of ordinary skill in the could readily identify relatedhomologs in various other species (e.g., mouse, rat, rabbit, dog,monkey, gibbon, chimp, ape, baboon, cow, pig, horse, sheep, cat andother species).

In addition, it should be clear that the present invention is notlimited to the specific nucleic acids disclosed herein. As discussedbelow, a “Killin gene” may contain a variety of different bases and yetstill produce a corresponding polypeptide that is functionallyindistinguishable from, and in some cases structurally identical to, thehuman gene disclosed herein.

Similarly, any reference to a nucleic acid should be read asencompassing a host cell containing that nucleic acid and, in somecases, capable of expressing the product of that nucleic acid. Inaddition to therapeutic considerations, cells expressing nucleic acidsof the present invention may prove useful in the context of screeningfor agents that induce, repress, inhibit, augment, interfere with,block, abrogate, stimulate or enhance the function of Killin.

A. Nucleic Acids Encoding Killin

Nucleic acids according to the present invention may encode an entireKillin gene, a domain of Killin that expresses a tumor suppressingfunction, or any other fragment of the Killin sequences set forthherein. The nucleic acid may be derived from genomic DNA, i.e., cloneddirectly from the genome of a particular organism. In preferredembodiments, however, the nucleic acid would comprise complementary DNA(cDNA). Also contemplated is a cDNA plus a natural intron or an intronderived from another gene; such engineered molecules are sometimereferred to as “mini-genes.” At a minimum, these and other nucleic acidsof the present invention may be used as molecular weight standards in,for example, gel electrophoresis.

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression or where non-coding regions such asintrons are to be targeted in an antisense strategy.

It also is contemplated that a given Killin from a given species may berepresented by natural variants that have slightly different nucleicacid sequences but, nonetheless, encode the same protein (see Table 1,above).

As used in this application, the term “a nucleic acid encoding a Killin”refers to a nucleic acid molecule that has been isolated free of totalcellular nucleic acid. In certain embodiments, the invention concerns anucleic acid sequence essentially as set forth in SEQ ID NO:2. The term“as set forth in SEQ ID NO:2” means that the nucleic acid sequencesubstantially corresponds to a portion of SEQ ID NO:2. The term“functionally equivalent codon” is used herein to refer to codons thatencode the same amino acid, such as the six codons for arginine orserine, and also refers to codons that encode biologically equivalentamino acids, as discussed in the following pages.

Allowing for the degeneracy of the genetic code, sequences that have atleast about 50%, usually at least about 60%, more usually about 70%,most usually about 80%, preferably at least about 90% and mostpreferably about 95% of nucleotides that are identical to thenucleotides of SEQ ID NO:2. Sequences that are essentially the same asthose set forth in SEQ ID NO:2 also may be functionally defined assequences that are capable of hybridizing to a nucleic acid segmentcontaining the complement of SEQ ID NO:2 under standard conditions.

The DNA segments of the present invention include those encodingbiologically functional equivalent Killin proteins and peptides, asdescribed above. Such sequences may arise as a consequence of codonredundancy and amino acid functional equivalency that are known to occurnaturally within nucleic acid sequences and the proteins thus encoded.Alternatively, functionally equivalent proteins or peptides may becreated via the application of recombinant DNA technology, in whichchanges in the protein structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by man may be introduced through the application ofsite-directed mutagenesis techniques or may be introduced randomly andscreened later for the desired function, as described below.

B. Oligonucleotide Probes and Primers

Naturally, the present invention also encompasses DNA segments that arecomplementary, or essentially complementary, to the sequence set forthin SEQ ID NO:2. Nucleic acid sequences that are “complementary” arethose that are capable of base-pairing according to the standardWatson-Crick complementary rules. As used herein, the term“complementary sequences” means nucleic acid sequences that aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of SEQ ID NO:2 under relativelystringent conditions such as those described herein. Such sequences mayencode the entire Killin protein or functional or non-functionalfragments thereof.

Alternatively, the hybridizing segments may be shorter oligonucleotides.Sequences of 17 bases long should occur only once in the human genomeand, therefore, suffice to specify a unique target sequence. Althoughshorter oligomers are easier to make and increase in vivo accessibility,numerous other factors are involved in determining the specificity ofhybridization. Both binding affinity and sequence specificity of anoligonucleotide to its complementary target increases with increasinglength. It is contemplated that exemplary oligonucleotides of 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100 or more base pairs will be used,although others are contemplated. Longer polynucleotides encoding 250,500, 1000, 1212, 1500, 2000, 2500, 3000 or longer are contemplated aswell. Such oligonucleotides will find use, for example, as probes inSouthern and Northern blots and as primers in amplification reactions.

Suitable hybridization conditions will be well known to those of skillin the art. In certain applications, for example, substitution of aminoacids by site-directed mutagenesis, it is appreciated that lowerstringency conditions are required. Under these conditions,hybridization may occur even though the sequences of probe and targetstrand are not perfectly complementary, but are mismatched at one ormore positions. Conditions may be rendered less stringent by increasingsalt concentration and decreasing temperature. For example, a mediumstringency condition could be provided by about 0.1 to 0.25 M NaCl attemperatures of about 37° C. to about 55° C., while a low stringencycondition could be provided by about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Thus,hybridization conditions can be readily manipulated, and thus willgenerally be a method of choice depending on the desired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 μM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C. Formamideand SDS also may be used to alter the hybridization conditions.

One method of using probes and primers of the present invention is inthe search for genes related to Killin or, more particularly, homologsof Killin from other species. Normally, the target DNA will be a genomicor cDNA library, although screening may involve analysis of RNAmolecules. By varying the stringency of hybridization, and the region ofthe probe, different degrees of homology may be discovered.

Another way of exploiting probes and primers of the present invention isin site-directed, or site-specific mutagenesis. Site-specificmutagenesis is a technique useful in the preparation of individualpeptides, or biologically functional equivalent proteins or peptides,through specific mutagenesis of the underlying DNA. The techniquefurther provides a ready ability to prepare and test sequence variants,incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

The technique typically employs a bacteriophage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage vectors are commercially available and their use isgenerally well known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double-strandedvector which includes within its sequence a DNA sequence encoding thedesired protein. An oligonucleotide primer bearing the desired mutatedsequence is synthetically prepared. This primer is then annealed withthe single-stranded DNA preparation, taking into account the degree ofmismatch when selecting hybridization conditions, and subjected to DNApolymerizing enzymes such as E. coli polymerase I Klenow fragment, inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cells,such as E. coli cells, and clones are selected that include recombinantvectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

C. Antisense Constructs

In some cases, mutant tumor suppressors may not be non-functional.Rather, they may have aberrant functions that cannot be overcome byreplacement gene therapy, even where the “wild-type” molecule isexpressed in amounts in excess of the mutant polypeptide. Antisensetreatments are one way of addressing this situation. Antisensetechnology also may be used to “knock-out” function of Killin in thedevelopment of cell lines or transgenic mice for research, diagnosticand screening purposes.

Antisense methodology takes advantage of the fact that nucleic acidstend to pair with “complementary” sequences. By complementary, it ismeant that polynucleotides are those which are capable of base-pairingaccording to the standard Watson-Crick complementarity rules. That is,the larger purines will base pair with the smaller pyrimidines to formcombinations of guanine paired with cytosine (G:C) and adenine pairedwith either thymine (A:T) in the case of DNA, or adenine paired withuracil (A:U) in the case of RNA. Inclusion of less common bases such asinosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others inhybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense polynucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription,

RNA processing, transport, translation and/or stability. Antisense RNAconstructs, or DNA encoding such antisense RNA's, may be employed toinhibit gene transcription or translation or both within a host cell,either in vitro or in vivo, such as within a host animal, including ahuman subject.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. It is contemplated that the most effective antisense constructswill include regions complementary to intron/exon splice junctions.Thus, it is proposed that a preferred embodiment includes an antisenseconstruct with complementarity to regions within 50-200 bases of anintron-exon splice junction. It has been observed that some exonsequences can be included in the construct without seriously affectingthe target selectivity thereof. The amount of exonic material includedwill vary depending on the particular exon and intron sequences used.One can readily test whether too much exon DNA is included simply bytesting the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

As stated above, “complementary” or “antisense” means polynucleotidesequences that are substantially complementary over their entire lengthand have very few base mismatches. For example, sequences of fifteenbases in length may be termed complementary when they have complementarynucleotides at thirteen or fourteen positions. Naturally, sequenceswhich are completely complementary will be sequences which are entirelycomplementary throughout their entire length and have no basemismatches. Other sequences with lower degrees of homology also arecontemplated. For example, an antisense construct which has limitedregions of high homology, but also contains a non-homologous region(e.g., ribozyme; see below) could be designed. These molecules, thoughhaving less than 50% homology, would bind to target sequences underappropriate conditions.

It may be advantageous to combine portions of genomic DNA with cDNA orsynthetic sequences to generate specific constructs. For example, wherean intron is desired in the ultimate construct, a genomic clone willneed to be used. The cDNA or a synthesized polynucleotide may providemore convenient restriction sites for the remaining portion of theconstruct and, therefore, would be used for the rest of the sequence.

D. Ribozymes

Another approach for addressing the “dominant negative” mutant tumorsuppressor is through the use of ribozymes. Although proteinstraditionally have been used for catalysis of nucleic acids, anotherclass of macromolecules has emerged as useful in this endeavor.Ribozymes are RNA-protein complexes that cleave nucleic acids in asite-specific fashion.

Ribozymes have specific catalytic domains that possess endonucleaseactivity (Kim and Cech, 1987; Gerlach et al., 1987; Forster and Symons,1987). For example, a large number of ribozymes accelerate phosphoestertransfer reactions with a high degree of specificity, often cleavingonly one of several phosphoesters in an oligonucleotide substrate (Cooket al., 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992).This specificity has been attributed to the requirement that thesubstrate bind via specific base-pairing interactions to the internalguide sequence (“IGS”) of the ribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cook et al., 1981). For example, U.S. Pat. No. 5,354,855reports that certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes. Thus, sequence-specificribozyme-mediated inhibition of gene expression may be particularlysuited to therapeutic applications (Scanlon et al., 1991; Sarver et al.,1990). Recently, it was reported that ribozymes elicited genetic changesin some cells lines to which they were applied; the altered genesincluded the oncogenes H-ras, c-fos and genes of HIV.

Most of this work involved the modification of a target mRNA, based on aspecific mutant codon that is cleaved by a specific ribozyme.

E. Vectors for Cloning, Gene Transfer and Expression

Within certain embodiments, expression vectors are employed to expressthe Killin polypeptide product, which can then be purified for varioususes. In other embodiments, the expression vectors are used in genetherapy. Expression requires that appropriate signals be provided in thevectors, and which include various regulatory elements, such asenhancers/promoters from both viral and mammalian sources that driveexpression of the genes of interest in host cells. Elements designed tooptimize messenger RNA stability and translatability in host cells alsoare defined. The conditions for the use of a number of dominant drugselection markers for establishing permanent, stable cell clonesexpressing the products are also provided, as is an element that linksexpression of the drug selection markers to expression of thepolypeptide.

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. In certain embodiments,expression includes both transcription of a gene and translation of mRNAinto a gene product. In other embodiments, expression only includestranscription of the nucleic acid encoding a gene of interest.

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques, which are described in Sambrook et al. (1989) and Ausubel etal. (1994), both incorporated herein by reference.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host organism. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

Regulatory Elements

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind such as RNA polymerase and other transcriptionfactors. The phrases “operatively positioned,” “operatively linked,”“under control,” and “under transcriptional control” mean that apromoter is in a correct functional location and/or orientation inrelation to a nucleic acid sequence to control transcriptionalinitiation and/or expression of that sequence. A promoter may or may notbe used in conjunction with an “enhancer,” which refers to a cis-actingregulatory sequence involved in the transcriptional activation of anucleic acid sequence.

A promoter may be one naturally-associated with a gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other prokaryotic, viral, or eukaryotic cell, andpromoters or enhancers not “naturally-occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. In addition to producing nucleicacid sequences of promoters and enhancers synthetically, sequences maybe produced using recombinant cloning and/or nucleic acid amplificationtechnology, including PCR™, in connection with the compositionsdisclosed herein (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906,each incorporated herein by reference). Furthermore, it is contemplatedthe control sequences that direct transcription and/or expression ofsequences within non-nuclear organelles such as mitochondria,chloroplasts, and the like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in the celltype, organelle, and organism chosen for expression. One example is thenative Killin promoter, set forth in SEQ ID NO: 3. Those of skill in theart of molecular biology generally know the use of promoters, enhancers,and cell type combinations for protein expression, for example, seeSambrook et al. (1989), incorporated herein by reference. The promotersemployed may be constitutive, tissue-specific, inducible, and/or usefulunder the appropriate conditions to direct high level expression of theintroduced DNA segment, such as is advantageous in the large-scaleproduction of recombinant proteins and/or peptides. The promoter may beheterologous or endogenous.

Table 2 lists several elements/promoters that may be employed, in thecontext of the present invention, to regulate the expression of a gene.This list is not intended to be exhaustive of all the possible elementsinvolved in the promotion of expression but, merely, to be exemplarythereof. Table 3 provides examples of inducible elements, which areregions of a nucleic acid sequence that can be activated in response toa specific stimulus.

TABLE 2 Promoter and/or Enhancer Promoter/Enhancer ReferencesImmunoglobulin Heavy Chain Banerji et al., 1983; Gilles et al., 1983;Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler et al.,1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al.;1990 Immunoglobulin Light Chain Queen et al., 1983; Picard et al., 1984T-Cell Receptor Luria et al., 1987; Winoto et al., 1989; Redondo et al.;1990 HLA DQ a and/or DQ β Sullivan et al., 1987 β-Interferon Goodbournet al., 1986; Fujita et al., 1987; Goodbourn et al., 1988 Interleukin-2Greene et al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin etal., 1990 MHC Class II 5 Koch et al., 1989 MHC Class II HLA-DRa Shermanet al., 1989 β-Actin Kawamoto et al., 1988; Ng et al.; 1989 MuscleCreatine Kinase (MCK) Jaynes et al., 1988; Horlick et al., 1989; Johnsonet al., 1989 Prealbumin (Transthyretin) Costa et al., 1988 Elastase IOmitz et al., 1987 Metallothionein (MTII) Karin et al., 1987; Culotta etal., 1989 Collagenase Pinkert et al., 1987; Angel et al., 1987 AlbuminPinkert et al., 1987; Tronche et al., 1989, 1990 α-Fetoprotein Godboutet al., 1988; Campere et al., 1989 t-Globin Bodine et al., 1987;Perez-Stable et al., 1990 β-Globin Trudel et al., 1987 c-fos Cohen etal., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985 Insulin Edlundet al., 1985 Neural Cell Adhesion Molecule Hirsh et al., 1990 (NCAM)α₁-Antitrypain Latimer et al., 1990 H2B (TH2B) Histone Hwang et al.,1990 Mouse and/or Type I Collagen Ripe et al., 1989 Glucose-RegulatedProteins Chang et al., 1989 (GRP94 and GRP78) Rat Growth Hormone Larsenet al., 1986 Human Serum Amyloid A (SAA) Edbrooke et al., 1989 TroponinI (TN I) Yutzey et al., 1989 Platelet-Derived Growth Factor Pech et al.,1989 (PDGF) Duchenne Muscular Dystrophy Klamut et al., 1990 SV40 Banerjiet al., 1981; Moreau et al., 1981; Sleigh et al., 1985; Firak et al.,1986; Herr et al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wanget al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al.,1988 Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980; Katinkaet al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; deVilliers et al., 1984; Hen et al., 1986; Satake et al., 1988; Campbelland/or Villarreal, 1988 Retroviruses Kriegler et al., 1982, 1983;Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze etal., 1986; Miksicek et al., 1986; Celander et al., 1987; Thiesen et al.,1988; Celander et al., 1988; Chol et al., 1988; Reisman et al., 1989Papilloma Virus Campo et al., 1983; Lusky et al., 1983; Spandidos and/orWilkie, 1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et al.,1987; Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987;Glue et al., 1988 Hepatitis B Virus Bulla et al., 1986; Jameel et al.,1986; Shaul et al., 1987; Spandau et al., 1988; Vannice et al., 1988Human Immunodeficiency Virus Muesing et al., 1987; Hauber et al., 1988;Jakobovits et al., 1988; Feng et al., 1988; Takebe et al., 1988; Rosenet al., 1988; Berkhout et al., 1989; Laspia et al., 1989; Sharp et al.,1989; Braddock et al., 1989 Cytomegalovirus (CMV) Weber et al., 1984;Boshart et al., 1985; Foecking et al., 1986 Gibbon Ape Leukemia VirusHolbrook et al., 1987; Quinn et al., 1989

TABLE 3 Inducible Elements Element Inducer References MT II PhorbolEster (TFA) Palmiter et al., 1982; Haslinger Heavy metals et al., 1985;Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin etal., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouse mammaryGlucocorticoids Huang et al., 1981; Lee et al., tumor virus) 1981;Majors et al., 1983; Chandler et al., 1983; Lee et al., 1984; Ponta etal., 1985; Sakai et al., 1988 β-Interferon poly(rI) × Tavernier et al.,1983 poly(rc) Adenovirus 5 E2 ElA Imperiale et al., 1984 CollagenasePhorbol Ester (TPA) Angel et al., 1987a Stromelysin Phorbol Ester (TPA)Angel et al., 1987b SV40 Phorbol Ester (TPA) Angel et al., 1987b MurineMX Gene Interferon, Newcastle Hug et al., 1988 Disease Virus GRP78 GeneA23187 Resendez et al., 1988 α-2-Macroglobulin IL-6 Kunz et al., 1989Vimentin Serum Rittling et al., 1989 MHC Class I Gene H-2κb InterferonBlanar et al., 1989 HSP70 ElA, SV40 Large T Taylor et al., 1989, 1990a,1990b Antigen Proliferin Phorbol Ester-TPA Mordacq et al., 1989 TumorNecrosis Factor PMA Hensel et al., 1989 Thyroid Stimulating ThyroidHormone Chatterjee et al., 1989 Hormone α Gene

The identity of tissue-specific promoters or elements, as well as assaysto characterize their activity, is well known to those of skill in theart. Examples of such regions include the human LIMK2 gene (Nomoto etal. 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), murineepididymal retinoic acid-binding gene (Lareyre et al., 1999), human CD4(Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen (Tsumaki, et al.,1998), D1A dopamine receptor gene (Lee, et al., 1997), insulin-likegrowth factor II (Wu et al., 1997), human platelet endothelial celladhesion molecule-1 (Almendro et al., 1996). Tumor specific promotersalso will find use in the present invention. Some such promoters are setforth in Tables 4 and 5.

TABLE 4 Candidate Tissue-Specific Promoters for Cancer Gene TherapyCancers in which promoter Normal cells in which Tissue-specific promoteris active promoter is active Carcinoembryonic antigen Most colorectalcarcinomas; Colonic mucosa; gastric (CEA)* 50% of lung carcinomas;40-50% mucosa; lung epithelia; of gastric carcinomas; eccrine sweatglands; cells in most pancreatic carcinomas; testes many breastcarcinomas Prostate-specific antigen Most prostate carcinomas Prostateepithelium (PSA) Vasoactive intestinal peptide Majority of non-smallcell Neurons; lymphocytes; mast (VIP) lung cancers cells; eosinophilsSurfactant protein A (SP-A) Many lung adenocarcinomas Type IIpneumocytes; Clara cells Human achaete-scute Most small cell lungcancers Neuroendocrine cells in lung homolog (hASH) Mucin-1 (MUC1)**Most adenocarcinomas Glandular epithelial cells in (originating from anytissue) breast and in respiratory, gastrointestinal, and genitourinarytracts Alpha-fetoprotein Most hepatocellular Hepatocytes (under certaincarcinomas; possibly many conditions); testis testicular cancers AlbuminMost hepatocellular Hepatocytes carcinomas Tyrosinase Most melanomasMelanocytes; astrocytes; Schwann cells; some neurons Tyrosine-bindingprotein Most melanomas Melanocytes; astrocytes, (TRP) Schwann cells;some neurons Keratin 14 Presumably many squamous Keratinocytes cellcarcinomas (e.g., Head and neck cancers) EBV LD-2 Many squamous cellKeratinocytes of upper carcinomas of head and neck digestiveKeratinocytes of upper digestive tract Glial fibrillary acidic proteinMany astrocytomas Astrocytes (GFAP) Myelin basic protein (MBP) Manygliomas Oligodendrocytes Testis-specific angiotensin- Possibly manytesticular Spermatazoa converting enzyme (Testis- cancers specific ACE)Osteocalcin Possibly many osteosarcomas Osteoblasts

TABLE 5 Candidate Promoters for Tissue-Specific Targeting of TumorsCancers in which Promoter Normal cells in which Promoter is activePromoter is active E2F-regulated promoter Almost all cancersProliferating cells HLA-G Many colorectal carcinomas; Lymphocytes;monocytes; many melanomas; possibly spermatocytes; trophoblast manyother cancers FasL Most melanomas; many Activated leukocytes: pancreaticcarcinomas; most neurons; endothelial cells; astrocytomas possibly manykeratinocytes; cells in other cancers immunoprivileged tissues; somecells in lungs, ovaries, liver, and prostate Myc-regulated promoter Mostlung carcinomas (both Proliferating cells (only some small cell andnon-small cell); cell-types): mammary most colorectal carcinomasepithelial cells (including non- proliferating) MAGE-1 Many melanomas;some non- Testis small cell lung carcinomas; some breast carcinomas VEGF70% of all cancers Cells at sites of (constitutive overexpression inneovascularization (but unlike many cancers) in tumors, expression istransient, less strong, and never constitutive) bFGF Presumably manydifferent Cells at sites of ischemia (but cancers, since bFGF unliketumors, expression is expression is induced by transient, less strong,and ischemic conditions never constitutive) COX-2 Most colorectalcarcinomas; Cells at sites of inflammation many lung carcinomas;possibly many other cancers IL-10 Most colorectal carcinomas; Leukocytesmany lung carcinomas; many squamous cell carcinomas of head and neck;possibly many other cancers GRP78/BiP Presumably many different Cells atsites of ishemia cancers, since GRP7S expression is induced bytumor-specific conditions CarG elements from Egr-1 Induced by ionizationCells exposed to ionizing radiation, so conceivably most radiation;leukocytes tumors upon irradiation

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

(ii) IRES

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′-methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, herein incorporated by reference).

(iii) Multi-Purpose Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector. See Carbonelli et al., 1999, Levenson et al., 1998,and Cocea, 1997, incorporated herein by reference. “Restriction enzymedigestion” refers to catalytic cleavage of a nucleic acid molecule withan enzyme that functions only at specific locations in a nucleic acidmolecule. Many of these restriction enzymes are commercially available.Use of such enzymes is widely understood by those of skill in the art.Frequently, a vector is linearized or fragmented using a restrictionenzyme that cuts within the MCS to enable exogenous sequences to beligated to the vector. “Ligation” refers to the process of formingphosphodiester bonds between two nucleic acid fragments, which may ormay not be contiguous with each other. Techniques involving restrictionenzymes and ligation reactions are well known to those of skill in theart of recombinant technology.

(iv) Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression (seeChandler et al., 1997, herein incorporated by reference.)

(v) Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and/or to minimize read through from thecassette into other sequences.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

(vi) Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and/or any suchsequence may be employed. Preferred embodiments include the SV40polyadenylation signal and/or the bovine growth hormone polyadenylationsignal, convenient and/or known to function well in various targetcells. Polyadenylation may increase the stability of the transcript ormay facilitate cytoplasmic transport.

(vii) Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

(viii) Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

(ix) Viral Vectors

The capacity of certain viral vectors to efficiently infect or entercells, to integrate into a host cell genome and stably express viralgenes, have led to the development and application of a number ofdifferent viral vector systems (Robbins et al., 1998). Viral systems arecurrently being developed for use as vectors for ex vivo and in vivogene transfer. For example, adenovirus, herpes-simplex virus, retrovirusand adeno-associated virus vectors are being evaluated currently fortreatment of diseases such as cancer, cystic fibrosis, Gaucher disease,renal disease and arthritis (Robbins and Ghivizzani, 1998; Imai et al.,1998; U.S. Pat. No. 5,670,488). The various viral vectors describedbelow, present specific advantages and disadvantages, depending on theparticular gene-therapeutic application.

Adenoviral Vectors. In particular embodiments, an adenoviral expressionvector is contemplated for the delivery of expression constructs.“Adenovirus expression vector” is meant to include those constructscontaining adenovirus sequences sufficient to (a) support packaging ofthe construct and (b) to ultimately express a tissue or cell-specificconstruct that has been cloned therein.

Adenoviruses comprise linear, double-stranded DNA, with a genome rangingfrom 30 to 35 kb in size (Reddy et al., 1998; Morrison et al., 1997;Chillon et al., 1999). An adenovirus expression vector according to thepresent invention comprises a genetically engineered form of theadenovirus. Advantages of adenoviral gene transfer include the abilityto infect a wide variety of cell types, including non-dividing cells, amid-sized genome, ease of manipulation, high infectivity and the abilityto be grown to high titers (Wilson, 1996). Further, adenoviral infectionof host cells does not result in chromosomal integration becauseadenoviral DNA can replicate in an episomal manner, without potentialgenotoxicity associated with other viral vectors. Adenoviruses also arestructurally stable (Marienfeld et al., 1999) and no genomerearrangement has been detected after extensive amplification (Parks etal., 1997; Bett et al., 1993).

Salient features of the adenovirus genome are an early region (E1, E2,E3 and E4 genes), an intermediate region (pIX gene, Iva2 gene), a lateregion (L1, L2, L3, L4 and L5 genes), a major late promoter (MLP),inverted-terminal-repeats (ITRs) and a yr sequence (Zheng, et al., 1999;Robbins et al., 1998; Graham and Prevec, 1995). The early genes E1, E2,E3 and E4 are expressed from the virus after infection and encodepolypeptides that regulate viral gene expression, cellular geneexpression, viral replication, and inhibition of cellular apoptosis.

Further on during viral infection, the MLP is activated, resulting inthe expression of the late (L) genes, encoding polypeptides required foradenovirus encapsidation. The intermediate region encodes components ofthe adenoviral capsid. Adenoviral inverted terminal repeats (ITRs;100-200 by in length), are cis elements, and function as origins ofreplication and are necessary for viral DNA replication. The yr sequenceis required for the packaging of the adenoviral genome.

A common approach for generating an adenoviruses for use as a genetransfer vector is the deletion of the E1 gene (E1), which is involvedin the induction of the E2, E3 and E4 promoters (Graham and Prevec,1995). Subsequently, a therapeutic gene or genes can be insertedrecombinantly in place of the El gene, wherein expression of thetherapeutic gene(s) is driven by the E1 promoter or a heterologouspromoter. The replication-deficient virus is then proliferated in a“helper” cell line that provides the E1 polypeptides in trans (e.g., thehuman embryonic kidney cell line 293). Thus, in the present invention itmay be convenient to introduce the transforming construct at theposition from which the El-coding sequences have been removed. However,the position of insertion of the construct within the adenovirussequences is not critical to the invention. Alternatively, the E3region, portions of the E4 region or both may be deleted, wherein aheterologous nucleic acid sequence under the control of a promoteroperable in eukaryotic cells is inserted into the adenovirus genome foruse in gene transfer (U.S. Pat. No. 5,670,488; U.S. Pat. No. 5,932,210,each specifically incorporated herein by reference).

Although adenovirus based vectors offer several unique advantages overother vector systems, they often are limited by vector immunogenicity,size constraints for insertion of recombinant genes and low levels ofreplication. The preparation of a recombinant adenovirus vector deletedof all open reading frames, comprising a full length dystrophin gene andthe terminal repeats required for replication (Haecker et al., 1997)offers some potentially promising advantages to the above mentionedadenoviral shortcomings. The vector was grown to high titer with ahelper virus in 293 cells and was capable of efficiently transducingdystrophin in mdx mice, in myotubes in vitro and muscle fibers in vivo.Helper-dependent viral vectors are discussed below.

A major concern in using adenoviral vectors is the generation of areplication-competent virus during vector production in a packaging cellline or during gene therapy treatment of an individual. The generationof a replication-competent virus could pose serious threat of anunintended viral infection and pathological consequences for thepatient. Armentano et al. (1990), describe the preparation of areplication-defective adenovirus vector, claimed to eliminate thepotential for the inadvertent generation of a replication-competentadenovirus (U.S. Pat. No. 5,824,544, specifically incorporated herein byreference). The replication-defective adenovirus method comprises adeleted E1 region and a relocated protein IX gene, wherein the vectorexpresses a heterologous, mammalian gene.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes and/or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus E1 region.Adenovirus growth and manipulation is known to those of skill in theart, and exhibits broad host range in vitro and in vivo (U.S. Pat. No.5,670,488; U.S. Pat. No. 5,932,210; U.S. Pat. No. 5,824,544). This groupof viruses can be obtained in high titers, e.g., 10⁹ to 10¹¹plaque-forming units per ml, and they are highly infective. The lifecycle of adenovirus does not require integration into the host cellgenome. The foreign genes delivered by adenovirus vectors are episomaland, therefore, have low genotoxicity to host cells. Many experiments,innovations, preclinical studies and clinical trials are currently underinvestigation for the use of adenoviruses as gene delivery vectors. Forexample, adenoviral gene delivery-based gene therapies are beingdeveloped for liver diseases (Han et al., 1999), psychiatric diseases(Lesch, 1999), neurological diseases (Smith, 1998; Hermens andVerhaagen, 1998), coronary diseases (Feldman et al., 1996), musculardiseases (Petrof, 1998), gastrointestinal diseases (Wu, 1998) andvarious cancers such as colorectal (Fujiwara and Tanaka, 1998; Dorai etal., 1999), pancreatic, bladder (Irie et al., 1999), head and neck(Blackwell et al., 1999), breast (Stewart et al., 1999), lung (Batra etal., 1999) and ovarian (Vanderkwaak et al., 1999).

Retroviral Vectors. In certain embodiments of the invention, the use ofretroviruses for gene delivery are contemplated. Retroviruses are RNAviruses comprising an RNA genome. When a host cell is infected by aretrovirus, the genomic RNA is reverse transcribed into a DNAintermediate which is integrated into the chromosomal DNA of infectedcells. This integrated DNA intermediate is referred to as a provirus. Aparticular advantage of retroviruses is that they can stably infectdividing cells with a gene of interest (e.g., a therapeutic gene) byintegrating into the host DNA, without expressing immunogenic viralproteins. Theoretically, the integrated retroviral vector will bemaintained for the life of the infected host cell, expressing the geneof interest.

The retroviral genome and the proviral DNA have three genes: gag, pol,and env, which are flanked by two long terminal repeat (LTR) sequences.The gag gene encodes the internal structural (matrix, capsid, andnucleocapsid) proteins; the pol gene encodes the RNA-directed DNApolymerase (reverse transcriptase) and the env gene encodes viralenvelope glycoproteins. The 5′ and 3′ LTRs serve to promotetranscription and polyadenylation of the virion RNAs. The LTR containsall other cis-acting sequences necessary for viral replication.

A recombinant retrovirus of the present invention may be geneticallymodified in such a way that some of the structural, infectious genes ofthe native virus have been removed and replaced instead with a nucleicacid sequence to be delivered to a target cell (U.S. Pat. No. 5,858,744;U.S. Pat. No. 5,739,018, each incorporated herein by reference). Afterinfection of a cell by the virus, the virus injects its nucleic acidinto the cell and the retrovirus genetic material can integrate into thehost cell genome. The transferred retrovirus genetic material is thentranscribed and translated into proteins within the host cell. As withother viral vector systems, the generation of a replication-competentretrovirus during vector production or during therapy is a majorconcern. Retroviral vectors suitable for use in the present inventionare generally defective retroviral vectors that are capable of infectingthe target cell, reverse transcribing their RNA genomes, and integratingthe reverse transcribed DNA into the target cell genome, but areincapable of replicating within the target cell to produce infectiousretroviral particles (e.g., the retroviral genome transferred into thetarget cell is defective in gag, the gene encoding virion structuralproteins, and/or in pol, the gene encoding reverse transcriptase). Thus,transcription of the provirus and assembly into infectious virus occursin the presence of an appropriate helper virus or in a cell linecontaining appropriate sequences enabling encapsidation withoutcoincident production of a contaminating helper virus.

The growth and maintenance of retroviruses is known in the art (U.S.Pat. No. 5,955,331; U.S. Pat. No. 5,888,502, each specificallyincorporated herein by reference). Nolan et al. describe the productionof stable high titre, helper-free retrovirus comprising a heterologousgene (U.S. Pat. No. 5,830,725, specifically incorporated herein byreference). Methods for constructing packaging cell lines useful for thegeneration of helper-free recombinant retroviruses with amphoteric orecotrophic host ranges, as well as methods of using the recombinantretroviruses to introduce a gene of interest into eukaryotic cells invivo and in vitro are contemplated in the present invention (U.S. Pat.No. 5,955,331).

Currently, the majority of all clinical trials for vector-mediated genedelivery use murine leukemia virus (MLV)-based retroviral vector genedelivery (Robbins et al., 1998; Miller et al., 1993). Disadvantages ofretroviral gene delivery includes a requirement for ongoing celldivision for stable infection and a coding capacity that prevents thedelivery of large genes. However, recent development of vectors such aslentivirus (e.g., HIV), simian immunodeficiency virus (SW) and equineinfectious-anemia virus (EIAV), which can infect certain non-dividingcells, potentially allow the in vivo use of retroviral vectors for genetherapy applications (Amado and Chen, 1999; Klimatcheva et al., 1999;White et al., 1999; Case et al., 1999). For example, HIV-based vectorshave been used to infect non-dividing cells such as neurons (Miyatake etal., 1999), islets (Leibowitz et al., 1999) and muscle cells (Johnstonet al., 1999). The therapeutic delivery of genes via retroviruses arecurrently being assessed for the treatment of various disorders such asinflammatory disease (Moldawer et al., 1999), AIDS (Amado et al., 1999;Engel and Kohn, 1999), cancer (Clay et al., 1999), cerebrovasculardisease (Weihl et al., 1999) and hemophilia (Kay, 1998).

Herpesviral Vectors. Herpes simplex virus (HSV) type I and type IIcontain a double-stranded, linear DNA genome of approximately 150 kb,encoding 70-80 genes. Wild type HSV are able to infect cells lyticallyand to establish latency in certain cell types (e.g., neurons). Similarto adenovirus, HSV also can infect a variety of cell types includingmuscle (Yeung et al., 1999), ear (Derby et al., 1999), eye (Kaufman etal., 1999), tumors (Yoon et al., 1999; Howard et al., 1999), lung (Kohutet al., 1998), neuronal (Garrido et al., 1999; Lachmann and Efstathiou,1999), liver (Miytake et al., 1999; Kooby et al., 1999) and pancreaticislets (Rabinovitch et al., 1999).

HSV viral genes are transcribed by cellular RNA polymerase II and aretemporally regulated, resulting in the transcription and subsequentsynthesis of gene products in roughly three discernable phases orkinetic classes. These phases of genes are referred to as the ImmediateEarly (IE) or alpha genes, Early (E) or beta genes and Late (L) or gammagenes. Immediately following the arrival of the genome of a virus in thenucleus of a newly infected cell, the IE genes are transcribed. Theefficient expression of these genes does not require prior viral proteinsynthesis. The products of IE genes are required to activatetranscription and regulate the remainder of the viral genome.

For use in therapeutic gene delivery, HSV must be renderedreplication-defective. Protocols for generating replication-defectiveHSV helper virus-free cell lines have been described (U.S. Pat. No.5,879,934; U.S. Pat. No. 5,851,826, each specifically incorporatedherein by reference in its entirety). One IE protein, Infected CellPolypeptide 4 (ICP4), also known as alpha 4 or Vmw175, is absolutelyrequired for both virus infectivity and the transition from IE to latertranscription. Thus, due to its complex, multifunctional nature andcentral role in the regulation of HSV gene expression, ICP4 hastypically been the target of HSV genetic studies.

Phenotypic studies of HSV viruses deleted of ICP4 indicate that suchviruses will be potentially useful for gene transfer purposes (Krisky etal., 1998a). One property of viruses deleted for ICP4 that makes themdesirable for gene transfer is that they only express the five other IEgenes: ICPO, ICP6, ICP27, ICP22 and ICP4? (DeLuca et al., 1985), withoutthe expression of viral genes encoding proteins that direct viral DNAsynthesis, as well as the structural proteins of the virus. Thisproperty is desirable for minimizing possible deleterious effects onhost cell metabolism or an immune response following gene transfer.Further deletion of IE genes ICP22 and ICP27, in addition to ICP4,substantially improve reduction of HSV cytotoxicity and prevented earlyand late viral gene expression (Krisky et al., 1998b).

The therapeutic potential of HSV in gene transfer has been demonstratedin various in vitro model systems and in vivo for diseases such asParkinson's (Yamada et al., 1999), retinoblastoma (Hayashi et al.,1999), intracerebral and intradermal tumors (Moriuchi et al., 1998),B-cell malignancies (Suzuki et al., 1998), ovarian cancer (Wang et al.,1998) and Duchenne muscular dystrophy (Huard et al., 1997).

Adeno-Associated Viral Vectors. Adeno-associated virus (AAV), a memberof the parvovirus family, is a human virus that is increasingly beingused for gene delivery therapeutics.

AAV has several advantageous features not found in other viral systems.First, AAV can infect a wide range of host cells, including non-dividingcells. Second, AAV can infect cells from different species. Third, AAVhas not been associated with any human or animal disease and does notappear to alter the biological properties of the host cell uponintegration. For example, it is estimated that 80-85% of the humanpopulation has been exposed to AAV. Finally, AAV is stable at a widerange of physical and chemical conditions which lends itself toproduction, storage and transportation requirements.

The AAV genome is a linear, single-stranded DNA molecule containing 4681nucleotides. The AAV genome generally comprises an internalnon-repeating genome flanked on each end by inverted terminal repeats(ITRs) of approximately 145 by in length. The ITRs have multiplefunctions, including origins of DNA replication, and as packagingsignals for the viral genome. The internal non-repeated portion of thegenome includes two large open reading frames, known as the AAVreplication (rep) and capsid (cap) genes. The rep and cap genes code forviral proteins that allow the virus to replicate and package the viralgenome into a virion. A family of at least four viral proteins areexpressed from the AAV rep region, Rep 78, Rep 68, Rep 52, and Rep 40,named according to their apparent molecular weight. The AAV cap regionencodes at least three proteins, VP1, VP2, and VP3.

AAV is a helper-dependent virus requiring co-infection with a helpervirus (e.g., adenovirus, herpesvirus or vaccinia) in order to form AAVvirions. In the absence of co-infection with a helper virus, AAVestablishes a latent state in which the viral genome inserts into a hostcell chromosome, but infectious virions are not produced. Subsequentinfection by a helper virus “rescues” the integrated genome, allowing itto replicate and package its genome into infectious AAV virions.Although AAV can infect cells from different species, the helper virusmust be of the same species as the host cell (e.g., human AAV willreplicate in canine cells co-infected with a canine adenovirus).

AAV has been engineered to deliver genes of interest by deleting theinternal non-repeating portion of the AAV genome and inserting aheterologous gene between the ITRs. The heterologous gene may befunctionally linked to a heterologous promoter (constitutive,cell-specific, or inducible) capable of driving gene expression intarget cells. To produce infectious recombinant AAV (rAAV) containing aheterologous gene, a suitable producer cell line is transfected with arAAV vector containing a heterologous gene. The producer cell isconcurrently transfected with a second plasmid harboring the AAV rep andcap genes under the control of their respective endogenous promoters orheterologous promoters. Finally, the producer cell is infected with ahelper virus.

Once these factors come together, the heterologous gene is replicatedand packaged as though it were a wild-type AAV genome. When target cellsare infected with the resulting rAAV virions, the heterologous geneenters and is expressed in the target cells. Because the target cellslack the rep and cap genes and the adenovirus helper genes, the rAAVcannot further replicate, package or form wild-type AAV.

The use of helper virus, however, presents a number of problems. First,the use of adenovirus in a rAAV production system causes the host cellsto produce both rAAV and infectious adenovirus. The contaminatinginfectious adenovirus can be inactivated by heat treatment (56° C. for 1hour). Heat treatment, however, results in approximately a 50% drop inthe titer of functional rAAV virions. Second, varying amounts ofadenovirus proteins are present in these preparations. For example,approximately 50% or greater of the total protein obtained in such rAAVvirion preparations is free adenovirus fiber protein. If not completelyremoved, these adenovirus proteins have the potential of eliciting animmune response from the patient. Third, AAV vector production methodswhich employ a helper virus require the use and manipulation of largeamounts of high titer infectious helper virus, which presents a numberof health and safety concerns, particularly in regard to the use of aherpesvirus. Fourth, concomitant production of helper virus particles inrAAV virion producing cells diverts large amounts of host cellularresources away from rAAV virion production, potentially resulting inlower rAAV virion yields.

Lentiviral Vectors. Lentiviruses are complex retroviruses, which, inaddition to the common retroviral genes gag, pol, and env, contain othergenes with regulatory or structural function. The higher complexityenables the virus to modulate its life cycle, as in the course of latentinfection. Some examples of lentivirus include the HumanImmunodeficiency Viruses: HIV-1, HIV-2 and the Simian ImmunodeficiencyVirus: SIV. Lentiviral vectors have been generated by multiplyattenuating the HIV virulence genes, for example, the genes env, vif,vpr, vpu and nef are deleted making the vector biologically safe.

Recombinant lentiviral vectors are capable of infecting non-dividingcells and can be used for both in vivo and ex vivo gene transfer andexpression of nucleic acid sequences. The lentiviral genome and theproviral DNA have the three genes found in retroviruses: gag, pol andenv, which are flanked by two long terminal repeat (LTR) sequences. Thegag gene encodes the internal structural (matrix, capsid andnucleocapsid) proteins; the pol gene encodes the RNA-directed DNApolymerase (reverse transcriptase), a protease and an integrase; and theenv gene encodes viral envelope glycoproteins. The 5′ and 3′ LTR's serveto promote transcription and polyadenylation of the virion RNA's. TheLTR contains all other cis-acting sequences necessary for viralreplication. Lentiviruses have additional genes including vif, vpr, tat,rev, vpu, nef and vpx.

Adjacent to the 5′ LTR are sequences necessary for reverse transcriptionof the genome (the tRNA primer binding site) and for efficientencapsidation of viral RNA into particles (the Psi site). If thesequences necessary for encapsidation (or packaging of retroviral RNAinto infectious virions) are missing from the viral genome, the cisdefect prevents encapsidation of genomic RNA. However, the resultingmutant remains capable of directing the synthesis of all virionproteins.

Lentiviral vectors are known in the art, see Naldini et al., (1996);Zufferey et al., (1997); U.S. Pat. Nos. 6,013,516;and 5,994,136. Ingeneral, the vectors are plasmid-based or virus-based, and areconfigured to carry the essential sequences for incorporating foreignnucleic acid, for selection and for transfer of the nucleic acid into ahost cell. The gag, pol and env genes of the vectors of interest alsoare known in the art. Thus, the relevant genes are cloned into theselected vector and then used to transform the target cell of interest.

Recombinant lentivirus capable of infecting a non-dividing cell whereina suitable host cell is transfected with two or more vectors carryingthe packaging functions, namely gag, pol and env, as well as rev andthat is described in U.S. Pat. No. 5,994,136, incorporated herein byreference. This describes a first vector that can provide a nucleic acidencoding a viral gag and a pol gene and another vector that can providea nucleic acid encoding a viral env to produce a packaging cell.Introducing a vector providing a heterologous gene, such as the STAT-1αgene in this invention, into that packaging cell yields a producer cellwhich releases infectious viral particles carrying the foreign gene ofinterest. The env preferably is an amphotropic envelope protein whichallows transduction of cells of human and other species.

One may target the recombinant virus by linkage of the envelope proteinwith an antibody or a particular ligand for targeting to a receptor of aparticular cell-type. By inserting a sequence (including a regulatoryregion) of interest into the viral vector, along with another gene whichencodes the ligand for a receptor on a specific target cell, forexample, the vector is now target-specific.

The vector providing the viral env nucleic acid sequence is associatedoperably with regulatory sequences, e.g., a promoter or enhancer. Theregulatory sequence can be any eukaryotic promoter or enhancer,including for example, the Moloney murine leukemia viruspromoter-enhancer element, the human cytomegalovirus enhancer or thevaccinia P7.5 promoter. In some cases, such as the Moloney murineleukemia virus promoter-enhancer element, the promoter-enhancer elementsare located within or adjacent to the LTR sequences.

The heterologous or foreign nucleic acid sequence, such as the STAT-1αencoding polynucleotide sequence herein, is linked operably to aregulatory nucleic acid sequence. Preferably, the heterologous sequenceis linked to a promoter, resulting in a chimeric gene. The heterologousnucleic acid sequence may also be under control of either the viral LTRpromoter-enhancer signals or of an internal promoter, and retainedsignals within the retroviral LTR can still bring about efficientexpression of the transgene. Marker genes may be utilized to assay forthe presence of the vector, and thus, to confirm infection andintegration. The presence of a marker gene ensures the selection andgrowth of only those host cells which express the inserts. Typicalselection genes encode proteins that confer resistance to antibioticsand other toxic substances, e.g., histidinol, puromycin, hygromycin,neomycin, methotrexate, etc., and cell surface markers.

The vectors are introduced via transfection or infection into thepackaging cell line. The packaging cell line produces viral particlesthat contain the vector genome. Methods for transfection or infectionare well known by those of skill in the art. After cotransfection of thepackaging vectors and the transfer vector to the packaging cell line,the recombinant virus is recovered from the culture media and titered bystandard methods used by those of skill in the art. Thus, the packagingconstructs can be introduced into human cell lines by calcium phosphatetransfection, lipofection or electroporation, generally together with adominant selectable marker, such as neo, DHFR, Gln synthetase or ADA,followed by selection in the presence of the appropriate drug andisolation of clones. The selectable marker gene can be linked physicallyto the packaging genes in the construct.

Lentiviral transfer vectors Naldini et al. (1996), have been used toinfect human cells growth-arrested in vitro and to transduce neuronsafter direct injection into the brain of adult rats. The vector wasefficient at transferring marker genes in vivo into the neurons and longterm expression in the absence of detectable pathology was achieved.Animals analyzed ten months after a single injection of the vectorshowed no decrease in the average level of transgene expression and nosign of tissue pathology or immune reaction (Blomer et al., 1997). Thus,in the present invention, one may graft or transplant cells infectedwith the recombinant lentivirus ex vivo, or infect cells in vivo.

Other Viral Vectors. The development and utility of viral vectors forgene delivery is constantly improving and evolving. Other viral vectorssuch as poxvirus; e.g., vaccinia virus (Gnant et al., 1999; Gnant etal., 1999), alpha virus; e.g., sindbis virus, Semliki forest virus(Lundstrom, 1999), reovirus (Coffey et al., 1998) and influenza A virus(Neumann et al., 1999) are contemplated for use in the present inventionand may be selected according to the requisite properties of the targetsystem.

In certain embodiments, vaccinia viral vectors are contemplated for usein the present invention. Vaccinia virus is a particularly usefuleukaryotic viral vector system for expressing heterologous genes. Forexample, when recombinant vaccinia virus is properly engineered, theproteins are synthesized, processed and transported to the plasmamembrane. Vaccinia viruses as gene delivery vectors have recently beendemonstrated to transfer genes to human tumor cells, e.g., EMAP-II(Gnant et al., 1999), inner ear (Derby et al., 1999), glioma cells,e.g., p53 (Timiryasova et al., 1999) and various mammalian cells, e.g.,P-450 (U.S. Pat. No. 5,506,138). The preparation, growth andmanipulation of vaccinia viruses are described in U.S. Pat. No.5,849,304 and U.S. Pat. No. 5,506,138 (each specifically incorporatedherein by reference).

In other embodiments, sindbis viral vectors are contemplated for use ingene delivery. Sindbis virus is a species of the alphavirus genus(Garoff and Li, 1998) which includes such important pathogens asVenezuelan, Western and Eastern equine encephalitis viruses (Sawai etal., 1999; Mastrangelo et al., 1999). In vitro, sindbis virus infects avariety of avian, mammalian, reptilian, and amphibian cells. The genomeof sindbis virus consists of a single molecule of single-stranded RNA,11,703 nucleotides in length. The genomic RNA is infectious, is cappedat the 5′ terminus and polyadenylated at the 3′ terminus, and serves asmRNA. Translation of a vaccinia virus 26S mRNA produces a polyproteinthat is cleaved co- and post-translationally by a combination of viraland presumably host-encoded proteases to give the three virus structuralproteins, a capsid protein (C) and the two envelope glycoproteins (E1and PE2, precursors of the virion E2).

Three features of sindbis virus suggest that it would be a useful vectorfor the expression of heterologous genes. First, its wide host range,both in nature and in the laboratory. Second, gene expression occurs inthe cytoplasm of the host cell and is rapid and efficient. Third,temperature-sensitive mutations in RNA synthesis are available that maybe used to modulate the expression of heterologous coding sequences bysimply shifting cultures to the non-permissive temperature at varioustime after infection. The growth and maintenance of sindbis virus isknown in the art (U.S. Pat. No. 5,217,879, specifically incorporatedherein by reference).

Chimeric Viral Vectors. Chimeric or hybrid viral vectors are beingdeveloped for use in therapeutic gene delivery and are contemplated foruse in the present invention. Chimeric poxviral/retroviral vectors(Holzer et al., 1999), adenoviral/retroviral vectors (Feng et al., 1997;Bilbao et al., 1997; Caplen et al., 1999) andadenoviral/adeno-associated viral vectors (Fisher et al., 1996; U.S.Pat. No. 5,871,982) have been described.

These “chimeric” viral gene transfer systems can exploit the favorablefeatures of two or more parent viral species. For example, Wilson etal., provide a chimeric vector construct which comprises a portion of anadenovirus, AAV 5′ and 3′ ITR sequences and a selected transgene,described below (U.S. Pat. No. 5,871,983, specifically incorporateherein by reference).

The adenovirus/AAV chimeric virus uses adenovirus nucleic acid sequencesas a shuttle to deliver a recombinant AAV/transgene genome to a targetcell. The adenovirus nucleic acid sequences employed in the hybridvector can range from a minimum sequence amount, which requires the useof a helper virus to produce the hybrid virus particle, to only selecteddeletions of adenovirus genes, which deleted gene products can besupplied in the hybrid viral production process by a selected packagingcell. At a minimum, the adenovirus nucleic acid sequences employed inthe pAdA shuttle vector are adenovirus genomic sequences from which allviral genes are deleted and which contain only those adenovirussequences required for packaging adenoviral genomic DNA into a preformedcapsid head. More specifically, the adenovirus sequences employed arethe cis-acting 5′ and 3′ inverted terminal repeat (ITR) sequences of anadenovirus (which function as origins of replication) and the native 5′packaging/enhancer domain, that contains sequences necessary forpackaging linear Ad genomes and enhancer elements for the E1 promoter.The adenovirus sequences may be modified to contain desired deletions,substitutions, or mutations, provided that the desired function is noteliminated.

The AAV sequences useful in the above chimeric vector are the viralsequences from which the rep and cap polypeptide encoding sequences aredeleted. More specifically, the AAV sequences employed are thecis-acting 5′ and 3′ inverted terminal repeat (ITR) sequences. Thesechimeras are characterized by high titer transgene delivery to a hostcell and the ability to stably integrate the transgene into the hostcell chromosome (U.S. Pat. No. 5,871,983, specifically incorporateherein by reference). In the hybrid vector construct, the AAV sequencesare flanked by the selected adenovirus sequences discussed above. The 5′and 3′ AAV ITR sequences themselves flank a selected transgene sequenceand associated regulatory elements, described below. Thus, the sequenceformed by the transgene and flanking 5′ and 3′ AAV sequences may beinserted at any deletion site in the adenovirus sequences of the vector.For example, the AAV sequences are desirably inserted at the site of thedeleted E1 a/E1b genes of the adenovirus. Alternatively, the AAVsequences may be inserted at an E3 deletion, E2a deletion, and so on. Ifonly the adenovirus 5′ ITR/packaging sequences and 3′ ITR sequences areused in the hybrid virus, the AAV sequences are inserted between them.

The transgene sequence of the vector and recombinant virus can be agene, a nucleic acid sequence or reverse transcript thereof,heterologous to the adenovirus sequence, which encodes a protein,polypeptide or peptide fragment of interest. The transgene isoperatively linked to regulatory components in a manner which permitstransgene transcription. The composition of the transgene sequence willdepend upon the use to which the resulting hybrid vector will be put.For example, one type of transgene sequence includes a therapeutic genewhich expresses a desired gene product in a host cell. These therapeuticgenes or nucleic acid sequences typically encode products foradministration and expression in a patient in vivo or ex vivo to replaceor correct an inherited or non-inherited genetic defect or treat anepigenetic disorder or disease.

(x) Non-Viral Transformation

Suitable methods for nucleic acid delivery for transformation of anorganelle, a cell, a tissue or an organism for use with the currentinvention are believed to include virtually any method by which anucleic acid (e.g., DNA) can be introduced into an organelle, a cell, atissue or an organism, as described herein or as would be known to oneof ordinary skill in the art. Such methods include, but are not limitedto, direct delivery of DNA such as by injection (U.S. Pat. Nos.5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932,5,656,610, 5,589,466 and 5,580,859, each incorporated herein byreference), including microinjection (Harland and Weintraub, 1985; U.S.Pat. No. 5,789,215, incorporated herein by reference); byelectroporation (U.S. Pat. No. 5,384,253, incorporated herein byreference); by calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE-dextranfollowed by polyethylene glycol (Gopal, 1985); by direct sonic loading(Fechheimer et al., 1987); by liposome mediated transfection (Nicolauand Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al.,1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectilebombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat.Nos. 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and5,538,880, and each incorporated herein by reference); by agitation withsilicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523and 5,464,765, each incorporated herein by reference); or byPEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S.Pat. Nos. 4,684,611 and 4,952,500, each incorporated herein byreference); by desiccation/inhibition-mediated DNA uptake (Potrykus etal., 1985). Through the application of techniques such as these,organelle(s), cell(s), tissue(s) or organism(s) may be stably ortransiently transformed.

Injection: In certain embodiments, a nucleic acid may be delivered to anorganelle, a cell, a tissue or an organism via one or more injections(i.e., a needle injection), such as, for example, either subcutaneously,intradermally, intramuscularly, intervenously or intraperitoneally.Methods of injection of vaccines are well known to those of ordinaryskill in the art (e.g., injection of a composition comprising a salinesolution). Further embodiments of the present invention include theintroduction of a nucleic acid by direct microinjection. Directmicroinjection has been used to introduce nucleic acid constructs intoXenopus oocytes (Harland and Weintraub, 1985).

Electroporation. In certain embodiments of the present invention, anucleic acid is introduced into an organelle, a cell, a tissue or anorganism via electroporation. Electroporation involves the exposure of asuspension of cells and DNA to a high-voltage electric discharge. Insome variants of this method, certain cell wall-degrading enzymes, suchas pectin-degrading enzymes, are employed to render the target recipientcells more susceptible to transformation by electroporation thanuntreated cells (U.S. Pat. No. 5,384,253, incorporated herein byreference). Alternatively, recipient cells can be made more susceptibleto transformation by mechanical wounding.

Transfection of eukaryotic cells using electroporation has been quitesuccessful. Mouse pre-B lymphocytes have been transfected with humankappa-immunoglobulin genes (Potter et al., 1984), and rat hepatocyteshave been transfected with the chloramphenicol acetyltransferase gene(Tur-Kaspa et al., 1986) in this manner.

To effect transformation by electroporation in cells such as, forexample, plant cells, one may employ either friable tissues, such as asuspension culture of cells or embryogenic callus or alternatively onemay transform immature embryos or other organized tissue directly. Inthis technique, one would partially degrade the cell walls of the chosencells by exposing them to pectin-degrading enzymes (pectolyases) ormechanically wounding in a controlled manner. Examples of some specieswhich have been transformed by electroporation of intact cells includemaize (U.S. Pat. No. 5,384,253; Rhodes et al., 1995; D′Halluin et al.,1992), wheat (Zhou et al., 1993), tomato (Hou and Lin, 1996), soybean(Christou et al., 1987) and tobacco (Lee et al., 1989).

One also may employ protoplasts for electroporation transformation ofplant cells (Bates, 1994; Lazzeri, 1995). For example, the generation oftransgenic soybean plants by electroporation of cotyledon-derivedprotoplasts is described by Dhir and Widholm in International PatentApplication No. WO 9217598, incorporated herein by reference. Otherexamples of species for which protoplast transformation has beendescribed include barley (Lazerri, 1995), sorghum (Battraw et al.,1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994) andtomato (Tsukada, 1989).

Calcium Phosphate. In other embodiments of the present invention, anucleic acid is introduced to the cells using calcium phosphateprecipitation. Human KB cells have been transfected with adenovirus 5DNA (Graham and Van Der Eb, 1973) using this technique. Also in thismanner, mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cellswere transfected with a neomycin marker gene (Chen and Okayama, 1987),and rat hepatocytes were transfected with a variety of marker genes(Rippe et al., 1990).

DEAE-Dextran: In another embodiment, a nucleic acid is delivered into acell using DEAE-dextran followed by polyethylene glycol. In this manner,reporter plasmids were introduced into mouse myeloma and erythroleukemiacells (Gopal, 1985).

Sonication Loading. Additional embodiments of the present inventioninclude the introduction of a nucleic acid by direct sonic loading. LTK⁻fibroblasts have been transfected with the thymidine kinase gene bysonication loading (Fechheimer et al., 1987).

Liposome-Mediated Transfection. In a further embodiment of theinvention, a nucleic acid may be entrapped in a lipid complex such as,for example, a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated is an nucleic acid complexed withLipofectamine (Gibco BRL) or Superfect (Qiagen).

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful (Nicolau and Sene, 1982; Fraley et al.,1979; Nicolau et al., 1987). The feasibility of liposome-mediateddelivery and expression of foreign DNA in cultured chick embryo, HeLaand hepatoma cells has also been demonstrated (Wong et al., 1980).

In certain embodiments of the invention, a liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, aliposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, a liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In other embodiments, a deliveryvehicle may comprise a ligand and a liposome.

Receptor Mediated Transfection: Still further, a nucleic acid may bedelivered to a target cell via receptor-mediated delivery vehicles.These take advantage of the selective uptake of macromolecules byreceptor-mediated endocytosis that will be occurring in a target cell.In view of the cell type-specific distribution of various receptors,this delivery method adds another degree of specificity to the presentinvention.

Certain receptor-mediated gene targeting vehicles comprise a cellreceptor-specific ligand and a nucleic acid-binding agent. Otherscomprise a cell receptor-specific ligand to which the nucleic acid to bedelivered has been operatively attached. Several ligands have been usedfor receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al.,1990; Perales et al., 1994; Myers, EPO 0273085), which establishes theoperability of the technique. Specific delivery in the context ofanother mammalian cell type has been described (Wu and Wu, 1993;incorporated herein by reference). In certain aspects of the presentinvention, a ligand will be chosen to correspond to a receptorspecifically expressed on the target cell population.

In other embodiments, a nucleic acid delivery vehicle component of acell-specific nucleic acid targeting vehicle may comprise a specificbinding ligand in combination with a liposome. The nucleic acid(s) to bedelivered are housed within the liposome and the specific binding ligandis functionally incorporated into the liposome membrane. The liposomewill thus specifically bind to the receptor(s) of a target cell anddeliver the contents to a cell. Such systems have been shown to befunctional using systems in which, for example, epidermal growth factor(EGF) is used in the receptor-mediated delivery of a nucleic acid tocells that exhibit upregulation of the EGF receptor.

In still further embodiments, the nucleic acid delivery vehiclecomponent of a targeted delivery vehicle may be a liposome itself, whichwill preferably comprise one or more lipids or glycoproteins that directcell-specific binding. For example, lactosyl-ceramide, agalactose-terminal asialganglioside, have been incorporated intoliposomes and observed an increase in the uptake of the insulin gene byhepatocytes (Nicolau et al., 1987). It is contemplated that thetissue-specific transforming constructs of the present invention can bespecifically delivered into a target cell in a similar manner.

F. Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with the present invention to producenucleic acid sequences, or their cognate polypeptides, proteins andpeptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986 and 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MAXBAC®2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROMCLONTECH®.

Other examples of expression systems include STRATAGENE®'S COMPLETECONTROL™ Inducible Mammalian Expression System, which involves asynthetic ecdysone-inducible receptor, or its pET Expression System, anE. coli expression system. Another example of an inducible expressionsystem is available from INVITROGEN®, which carries the T-REx™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

Primary mammalian cell cultures may be prepared in various ways. Inorder for the cells to be kept viable while in vitro and in contact withthe expression construct, it is necessary to ensure that the cellsmaintain contact with the correct ratio of oxygen and carbon dioxide andnutrients but are protected from microbial contamination. Cell culturetechniques are well documented.

One embodiment of the foregoing involves the use of gene transfer toimmortalize cells for the production of proteins. The gene for theprotein of interest may be transferred as described above intoappropriate host cells followed by culture of cells under theappropriate conditions. The gene for virtually any polypeptide may beemployed in this manner. The generation of recombinant expressionvectors, and the elements included therein, are discussed above.Alternatively, the protein to be produced may be an endogenous proteinnormally synthesized by the cell in question.

Examples of useful mammalian host cell lines are Vero and HeLa cells andcell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2,NIH3T3, RIN and MDCK cells. In addition, a host cell strain may bechosen that modulates the expression of the inserted sequences, ormodifies and process the gene product in the manner desired. Suchmodifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to insure the correctmodification and processing of the foreign protein expressed.

A number of selection systems may be used including, but not limited to,HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase andadenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt-cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to; gpt, that confersresistance to mycophenolic acid; neo, that confers resistance to theaminoglycoside G418; and hygro, that confers resistance to hygromycin.

G. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organisms that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny.

Host cells may be derived from prokaryotes or eukaryotes, depending uponwhether the desired result is replication of the vector or expression ofpart or all of the vector-encoded nucleic acid sequences. Numerous celllines and cultures are available for use as a host cell, and they can beobtained through the American Type Culture Collection (ATCC), which isan organization that serves as an archive for living cultures andgenetic materials (www.atcc.org). An appropriate host can be determinedby one of skill in the art based on the vector backbone and the desiredresult. A plasmid or cosmid, for example, can be introduced into aprokaryote host cell for replication of many vectors. Bacterial cellsused as host cells for vector replication and/or expression includeDH5α, JM109, and KC8, as well as a number of commercially availablebacterial hosts such as SURE® Competent Cells and SOLOPACK™ Gold Cells(STRATAGENE®, La Jolla). Alternatively, bacterial cells such as E. coliLE392 could be used as host cells for phage viruses.

Examples of eukaryotic host cells for replication and/or expression of avector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Manyhost cells from various cell types and organisms are available and wouldbe known to one of skill in the art. Similarly, a viral vector may beused in conjunction with either a eukaryotic or prokaryotic host cell,particularly one that is permissive for replication or expression of thevector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

H. Cell Propagation

Animal cells can be propagated in vitro in two modes: as non-anchoragedependent cells growing in suspension throughout the bulk of the cultureor as anchorage-dependent cells requiring attachment to a solidsubstrate for their propagation (i.e., a monolayer type of cell growth).Non-anchorage dependent or suspension cultures from continuousestablished cell lines are the most widely used means of large scaleproduction of cells and cell products. However, suspension culturedcells have limitations, such as tumorigenic potential and lower proteinproduction than adherent T-cells.

Large scale suspension culture of mammalian cells in stirred tanks is acommon method for production of recombinant proteins. Two suspensionculture reactor designs are in wide use—the stirred reactor and theairlift reactor. The stirred design has successfully been used on an8000 liter capacity for the production of interferon. Cells are grown ina stainless steel tank with a height-to-diameter ratio of 1:1 to 3:1.The culture is usually mixed with one or more agitators, based on bladeddisks or marine propeller patterns. Agitator systems offering less shearforces than blades have been described. Agitation may be driven eitherdirectly or indirectly by magnetically coupled drives. Indirect drivesreduce the risk of microbial contamination through seals on stirrershafts.

The airlift reactor, also initially described for microbial fermentationand later adapted for mammalian culture, relies on a gas stream to bothmix and oxygenate the culture. The gas stream enters a riser section ofthe reactor and drives circulation. Gas disengages at the culturesurface, causing denser liquid free of gas bubbles to travel downward inthe downcomer section of the reactor. The main advantage of this designis the simplicity and lack of need for mechanical mixing. Typically, theheight-to-diameter ratio is 10:1. The airlift reactor scales uprelatively easily, has good mass transfer of gases and generatesrelatively low shear forces.

The antibodies of the present invention are particularly useful for theisolation of antigens by immunoprecipitation. Immunoprecipitationinvolves the separation of the target antigen component from a complexmixture, and is used to discriminate or isolate minute amounts ofprotein. For the isolation of membrane proteins cells must besolubilized into detergent micelles. Non-ionic salts are preferred,since other agents such as bile salts, precipitate at acid pH or in thepresence of bivalent cations. Antibodies are and their uses arediscussed further, below.

III. Generating Antibodies Reactive with Killin

In another aspect, the present invention contemplates an antibody thatis immunoreactive with a Killin molecule of the present invention, orany portion thereof. An antibody can be a polyclonal or a monoclonalantibody. In a preferred embodiment, an antibody is a monoclonalantibody. Means for preparing and characterizing antibodies are wellknown in the art (see, e.g., Harlow and Lane, 1988).

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a polypeptide of the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically an animalused for production of anti-antisera is a non-human animal includingrabbits, mice, rats, hamsters, pigs or horses. Because of the relativelylarge blood volume of rabbits, a rabbit is a preferred choice forproduction of polyclonal antibodies.

Antibodies, both polyclonal and monoclonal, specific for isoforms ofantigen may be prepared using conventional immunization techniques, aswill be generally known to those of skill in the art. A compositioncontaining antigenic epitopes of the compounds of the present inventioncan be used to immunize one or more experimental animals, such as arabbit or mouse, which will then proceed to produce specific antibodiesagainst the compounds of the present invention. Polyclonal antisera maybe obtained, after allowing time for antibody generation, simply bybleeding the animal and preparing serum samples from the whole blood.

It is proposed that the monoclonal antibodies of the present inventionwill find useful application in standard immunochemical procedures, suchas ELISA and Western blot methods and in immunohistochemical proceduressuch as tissue staining, as well as in other procedures which mayutilize antibodies specific to Killin-related antigen epitopes.Additionally, it is proposed that monoclonal antibodies specific to theparticular Killin of different species may be utilized in other usefulapplications

In general, both polyclonal and monoclonal antibodies against Killin maybe used in a variety of embodiments. For example, they may be employedin antibody cloning protocols to obtain cDNAs or genes encoding otherKillin. They may also be used in inhibition studies to analyze theeffects of Killin related peptides in cells or animals. Anti-Killinantibodies will also be useful in immunolocalization studies to analyzethe distribution of Killin during various cellular events, for example,to determine the cellular or tissue-specific distribution of Killinpolypeptides under different points in the cell cycle. A particularlyuseful application of such antibodies is in purifying native orrecombinant Killin, for example, using an antibody affinity column. Theoperation of all such immunological techniques will be known to those ofskill in the art in light of the present disclosure.

Means for preparing and characterizing antibodies are well known in theart (see, e.g., Harlow and Lane, 1988; incorporated herein byreference). More specific examples of monoclonal antibody preparationare give in the examples below.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As also is well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified Killin protein, polypeptide or peptide or cellexpressing high levels of Killin. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells. Rodents such as mice and rats are preferred animals, however, theuse of rabbit, sheep frog cells is also possible. The use of rats mayprovide certain advantages (Goding, 1986), but mice are preferred, withthe BALB/c mouse being most preferred as this is most routinely used andgenerally gives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B-lymphocytes (B-cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, 1986; Campbell, 1984). For example, wherethe immunized animal is a mouse, one may use P3-X63/Ag8, P3-X63-Ag8.653,NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 andS194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all usefulin connection with cell fusions.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, by Gefter et al., (1977). The use of electricallyinduced fusion methods is also appropriate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,around 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

IV. Diagnosing Cancers Involving Killin

Killin and the corresponding gene may be employed as a diagnostic orprognostic indicator of cancer. More specifically, point mutations,deletions, insertions or regulatory pertubations relating to Killin maycause cancer or promote cancer development, cause or promoter tumorprogression at a primary site, and/or cause or promote metastasis. Otherphenomena associated with malignancy that may be affected by Killinexpression include angiogenesis and tissue invasion.

A. Genetic Diagnosis

One embodiment of the instant invention comprises a method for detectingvariation in the expression of Killin. This may comprises determiningthat level of Killin or determining specific alterations in theexpressed product. Obviously, this sort of assay has importance in thediagnosis of related cancers. Such cancer may involve cancers of thebrain (glioblastomas, medulloblastoma, astrocytoma, oligodendroglioma,ependymomas), lung, liver, spleen, kidney, pancreas, small intestine,blood cells, lymph node, colon, breast, endometrium, stomach, prostate,testicle, ovary, skin, head and neck, esophagus, bone marrow, blood orother tissue. In particular, the present invention relates to thediagnosis of gliomas.

The biological sample can be any tissue or fluid. Various embodimentsinclude cells of the skin, muscle, facia, brain, prostate, breast,endometrium, lung, head & neck, pancreas, small intestine, blood cells,liver, testes, ovaries, colon, skin, stomach, esophagus, spleen, lymphnode, bone marrow or kidney. Other embodiments include fluid samplessuch as peripheral blood, lymph fluid, ascites, serous fluid, pleuraleffusion, sputum, cerebrospinal fluid, lacrimal fluid, stool or urine.

Nucleic acid used is isolated from cells contained in the biologicalsample, according to standard methodologies (Sambrook et al., 1989). Thenucleic acid may be genomic DNA or fractionated or whole cell RNA. WhereRNA is used, it may be desired to convert the RNA to a complementaryDNA. In one embodiment, the RNA is whole cell RNA; in another, it ispoly-A RNA. Normally, the nucleic acid is amplified.

Depending on the format, the specific nucleic acid of interest isidentified in the sample directly using amplification or with a second,known nucleic acid following amplification. Next, the identified productis detected. In certain applications, the detection may be performed byvisual means (e.g., ethidium bromide staining of a gel). Alternatively,the detection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of radiolabel or fluorescentlabel or even via a system using electrical or thermal impulse signals(Affymax Technology; Bellus, 1994).

Following detection, one may compare the results seen in a given patientwith a statistically significant reference group of normal patients andpatients that have Killin-related pathologies. In this way, it ispossible to correlate the amount or kind of Killin detected with variousclinical states.

Various types of defects have been identified by the present inventors.Thus, “alterations” should be read as including deletions, insertions,point mutations and duplications. Point mutations result in stop codons,frameshift mutations or amino acid substitutions. Somatic mutations arethose occurring in non-germline tissues. Germ-line tissue can occur inany tissue and are inherited. Mutations in and outside the coding regionalso may affect the amount of Killin produced, both by altering thetranscription of the gene or in destabilizing or otherwise altering theprocessing of either the transcript (mRNA) or protein.

A cell takes a genetic step toward oncogenic transformation when oneallele of a tumor suppressor gene is inactivated due to inheritance of agermline lesion or acquisition of a somatic mutation. The inactivationof the other allele of the gene usually involves a somatic micromutationor chromosomal allelic deletion that results in loss of heterozygosity(LOH). Alternatively, both copies of a tumor suppressor gene may be lostby homozygous deletion.

It is contemplated that other mutations in the Killin gene may beidentified in accordance with the present invention. A variety ofdifferent assays are contemplated in this regard, including but notlimited to, fluorescent in situ hybridization (FISH), direct DNAsequencing, PFGE analysis, Southern or Northern blotting,single-stranded conformation analysis (SSCA), RNAse protection assay,allele-specific oligonucleotide (ASO), dot blot analysis, denaturinggradient gel electrophoresis, RFLP and PCR™-SSCP.

(i) Primers and Probes

The term primer, as defined herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty base pairs in length, but longer sequences can beemployed. Primers may be provided in double-stranded or single-strandedform, although the single-stranded form is preferred. Probes are defineddifferently, although they may act as primers. Probes, while perhapscapable of priming, are designed to binding to the target DNA or RNA andneed not be used in an amplification process. In particular embodiments,the probes or primers are labeled with radioactive species (³²P, ¹⁴C,³⁵S, ³H, or other label), with a fluorophore (rhodamine, fluorescein) ora chemillumiscent (luciferase).

(ii) Template Dependent Amplification Methods

A number of template dependent processes are available to amplify themarker sequences present in a given template sample. One of the bestknown amplification methods is the polymerase chain reaction (referredto as PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159, and in Innis et al., 1990, each of which isincorporated herein by reference in its entirety.

Briefly, in PCR™, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated.

A reverse transcriptase PCR™ amplification procedure may be performed inorder to quantify the amount of mRNA amplified. Methods of reversetranscribing RNA into cDNA are well known and described in Sambrook etal., 1989. Alternative methods for reverse transcription utilizethermostable, RNA-dependent DNA polymerases. These methods are describedin WO 90/07641 filed Dec. 21, 1990. Polymerase chain reactionmethodologies are well known in the art.

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in EPO No. 320 308, incorporated herein by reference in itsentirety. In LCR, two complementary probe pairs are prepared, and in thepresence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR™, bound ligated units dissociate from thetarget and then serve as “target sequences” for ligation of excess probepairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR forbinding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, mayalso be used as still another amplification method in the presentinvention. In this method, a replicative sequence of RNA that has aregion complementary to that of a target is added to a sample in thepresence of an RNA polymerase. The polymerase will copy the replicativesequence that can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention, Walker et al., (1992).

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e., nick translation. Asimilar method, called Repair Chain Reaction (RCR), involves annealingseveral probes throughout a region targeted for amplification, followedby a repair reaction in which only two of the four bases are present.The other two bases can be added as biotinylated derivatives for easydetection. A similar approach is used in SDA. Target specific sequencescan also be detected using a cyclic probe reaction (CPR). In CPR, aprobe having 3′ and 5′ sequences of non-specific DNA and a middlesequence of specific RNA is hybridized to DNA that is present in asample. Upon hybridization, the reaction is treated with RNase H, andthe products of the probe identified as distinctive products that arereleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated.

Still another amplification methods described in GB Application No. 2202 328, and in PCT Application No. PCT/US89/01025, each of which isincorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,“modified” primers are used in a PCR™-like, template- andenzyme-dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes are added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989; Gingeras et al., PCTApplication WO 88/10315, incorporated herein by reference in theirentirety). In NASBA, the nucleic acids can be prepared for amplificationby standard phenol/chloroform extraction, heat denaturation of aclinical sample, treatment with lysis buffer and minispin columns forisolation of DNA and RNA or guanidinium chloride extraction of RNA.These amplification techniques involve annealing a primer which hastarget specific sequences. Following polymerization, DNA/RNA hybrids aredigested with RNase H while double stranded DNA molecules are heatdenatured again. In either case the single stranded DNA is made fullydouble-stranded by addition of second target specific primer, followedby polymerization. The double-stranded DNA molecules are then multiplytranscribed by an RNA polymerase such as T7 or SP6. In an isothermalcyclic reaction, the RNA's are reverse transcribed into single-strandedDNA, which is then converted to double stranded DNA, and thentranscribed once again with an RNA polymerase such as T7 or SP6. Theresulting products, whether truncated or complete, indicate targetspecific sequences.

Davey et al., EPO No. 329 822 (incorporated herein by reference in itsentirety) disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H (RNase H, anRNase specific for RNA in duplex with either DNA or RNA). The resultantssDNA is a template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting in a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

Miller et al., PCT Application WO 89/06700 (incorporated herein byreference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic, i.e., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “RACE” and “one-sidedPCR™” (Frohman, 1990; Ohara et al., 1989; each herein incorporated byreference in their entirety).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention. Wu etal., (1989), incorporated herein by reference in its entirety.

(iii) Southern/Northern Blotting

Blotting techniques are well known to those of skill in the art.Southern blotting involves the use of DNA as a target, whereas Northernblotting involves the use of RNA as a target. Each provide differenttypes of information, although cDNA blotting is analogous, in manyaspects, to blotting or RNA species.

Briefly, a probe is used to target a DNA or RNA species that has beenimmobilized on a suitable matrix, often a filter of nitrocellulose. Thedifferent species should be spatially separated to facilitate analysis.This often is accomplished by gel electrophoresis of nucleic acidspecies followed by “blotting” on to the filter.

Subsequently, the blotted target is incubated with a probe (usuallylabeled) under conditions that promote denaturation and rehybridization.Because the probe is designed to base pair with the target, the probewill binding a portion of the target sequence under renaturingconditions. Unbound probe is then removed, and detection is accomplishedas described above.

(iv) Separation Methods

It normally is desirable, at one stage or another, to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification has occurred. Inone embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods. See Sambrook et al., 1989.

Alternatively, chromatographic techniques may be employed to effectseparation. There are many kinds of chromatography which may be used inthe present invention: adsorption, partition, ion-exchange and molecularsieve, and many specialized techniques for using them including column,paper, thin-layer and gas chromatography (Freifelder, 1982).

(v) Detection Methods

Products may be visualized in order to confirm amplification of themarker sequences. One typical visualization method involves staining ofa gel with ethidium bromide and visualization under UV light.Alternatively, if the amplification products are integrally labeled withradio- or fluorometrically-labeled nucleotides, the amplificationproducts can then be exposed to x-ray film or visualized under theappropriate stimulating spectra, following separation.

In one embodiment, visualization is achieved indirectly. Followingseparation of amplification products, a labeled nucleic acid probe isbrought into contact with the amplified marker sequence. The probepreferably is conjugated to a chromophore but may be radiolabeled. Inanother embodiment, the probe is conjugated to a binding partner, suchas an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

In one embodiment, detection is by a labeled probe. The techniquesinvolved are well known to those of skill in the art and can be found inmany standard books on molecular protocols. See Sambrook et al., 1989.For example, chromophore or radiolabel probes or primers identify thetarget during or following amplification.

One example of the foregoing is described in U.S. Pat. No. 5,279,721,incorporated by reference herein, which discloses an apparatus andmethod for the automated electrophoresis and transfer of nucleic acids.The apparatus permits electrophoresis and blotting without externalmanipulation of the gel and is ideally suited to carrying out methodsaccording to the present invention.

In addition, the amplification products described above may be subjectedto sequence analysis to identify specific kinds of variations usingstandard sequence analysis techniques. Within certain methods,exhaustive analysis of genes is carried out by sequence analysis usingprimer sets designed for optimal sequencing (Pignon et al., 1994). Thepresent invention provides methods by which any or all of these types ofanalyses may be used. Using the sequences disclosed herein,oligonucleotide primers may be designed to permit the amplification ofsequences throughout the Killin gene that may then be analyzed by directsequencing.

(vi) Kit Components

All the essential materials and reagents required for detecting andsequencing KILLIN and variants thereof may be assembled together in akit. This generally will comprise preselected primers and probes. Alsoincluded may be enzymes suitable for amplifying nucleic acids includingvarious polymerases (RT, Taq, Sequenase™ etc.), deoxynucleotides andbuffers to provide the necessary reaction mixture for amplification.Such kits also generally will comprise, in suitable means, distinctcontainers for each individual reagent and enzyme as well as for eachprimer or probe.

(vii) Design and Theoretical Considerations for Relative QuantitativeRT-PCR™

Reverse transcription (RT) of RNA to cDNA followed by relativequantitative PCR™ (RT-PCR™) can be used to determine the relativeconcentrations of specific mRNA species isolated from patients. Bydetermining that the concentration of a specific mRNA species varies, itis shown that the gene encoding the specific mRNA species isdifferentially expressed.

In PCR™, the number of molecules of the amplified target DNA increase bya factor approaching two with every cycle of the reaction until somereagent becomes limiting. Thereafter, the rate of amplification becomesincreasingly diminished until there is no increase in the amplifiedtarget between cycles. If a graph is plotted in which the cycle numberis on the X axis and the log of the concentration of the amplifiedtarget DNA is on the Y axis, a curved line of characteristic shape isformed by connecting the plotted points. Beginning with the first cycle,the slope of the line is positive and constant. This is said to be thelinear portion of the curve. After a reagent becomes limiting, the slopeof the line begins to decrease and eventually becomes zero. At thispoint the concentration of the amplified target DNA becomes asymptoticto some fixed value. This is said to be the plateau portion of thecurve.

The concentration of the target DNA in the linear portion of the PCR™amplification is directly proportional to the starting concentration ofthe target before the reaction began. By determining the concentrationof the amplified products of the target DNA in PCR™ reactions that havecompleted the same number of cycles and are in their linear ranges, itis possible to determine the relative concentrations of the specifictarget sequence in the original DNA mixture. If the DNA mixtures arecDNAs synthesized from RNAs isolated from different tissues or cells,the relative abundances of the specific mRNA from which the targetsequence was derived can be determined for the respective tissues orcells. This direct proportionality between the concentration of the PCR™products and the relative mRNA abundances is only true in the linearrange of the PCR™ reaction.

The final concentration of the target DNA in the plateau portion of thecurve is determined by the availability of reagents in the reaction mixand is independent of the original concentration of target DNA.Therefore, the first condition that must be met before the relativeabundances of a mRNA species can be determined by RT-PCR™ for acollection of RNA populations is that the concentrations of theamplified PCR™ products must be sampled when the PCR™ reactions are inthe linear portion of their curves.

The second condition that must be met for an RT-PCR™ experiment tosuccessfully determine the relative abundances of a particular mRNAspecies is that relative concentrations of the amplifiable cDNAs must benormalized to some independent standard. The goal of an RT-PCR™experiment is to determine the abundance of a particular mRNA speciesrelative to the average abundance of all mRNA species in the sample. Inthe experiments described below, mRNAs for β-actin, asparaginesynthetase and lipocortin II were used as external and internalstandards to which the relative abundance of other mRNAs are compared.

Most protocols for competitive PCR™ utilize internal PCR™ standards thatare approximately as abundant as the target. These strategies areeffective if the products of the PCR™ amplifications are sampled duringtheir linear phases. If the products are sampled when the reactions areapproaching the plateau phase, then the less abundant product becomesrelatively over represented. Comparisons of relative abundances made formany different RNA samples, such as is the case when examining RNAsamples for differential expression, become distorted in such a way asto make differences in relative abundances of RNAs appear less than theyactually are. This is not a significant problem if the internal standardis much more abundant than the target. If the internal standard is moreabundant than the target, then direct linear comparisons can be madebetween RNA samples.

The above discussion describes theoretical considerations for an RT-PCR™assay for clinically derived materials. The problems inherent inclinical samples are that they are of variable quantity (makingnormalization problematic), and that they are of variable quality(necessitating the co-amplification of a reliable internal control,preferably of larger size than the target). Both of these problems areovercome if the RT-PCR™ is performed as a relative quantitative RT-PCR™with an internal standard in which the internal standard is anamplifiable cDNA fragment that is larger than the target cDNA fragmentand in which the abundance of the mRNA encoding the internal standard isroughly 5-100 fold higher than the mRNA encoding the target. This assaymeasures relative abundance, not absolute abundance of the respectivemRNA species.

Other studies may be performed using a more conventional relativequantitative RT-PCR™ assay with an external standard protocol. Theseassays sample the PCR™ products in the linear portion of theiramplification curves. The number of PCR™ cycles that are optimal forsampling must be empirically determined for each target cDNA fragment.In addition, the reverse transcriptase products of each RNA populationisolated from the various tissue samples must be carefully normalizedfor equal concentrations of amplifiable cDNAs. This consideration isvery important since the assay measures absolute mRNA abundance.Absolute mRNA abundance can be used as a measure of differential geneexpression only in normalized samples. While empirical determination ofthe linear range of the amplification curve and normalization of cDNApreparations are tedious and time consuming processes, the resultingRT-PCR™ assays can be superior to those derived from the relativequantitative RT-PCR™ assay with an internal standard.

One reason for this advantage is that without the internalstandard/competitor, all of the reagents can be converted into a singlePCR™ product in the linear range of the amplification curve, thusincreasing the sensitivity of the assay. Another reason is that withonly one PCR™ product, display of the product on an electrophoretic gelor another display method becomes less complex, has less background andis easier to interpret.

(viii) Chip Technologies

Specifically contemplated by the present inventors are chip-based DNAtechnologies such as those described by Hacia et al. (1996) andShoemaker et al. (1996). Briefly, these techniques involve quantitativemethods for analyzing large numbers of genes rapidly and accurately. Bytagging genes with oligonucleotides or using fixed probe arrays, one canemploy chip technology to segregate target molecules as high densityarrays and screen these molecules on the basis of hybridization. Seealso Pease et al. (1994); Fodor et al. (1991).

B. Immunodiagnosis

Antibodies of the present invention can be used in characterizing theKillin content of healthy and diseased tissues, through techniques suchas ELISAs and Western blotting. This may provide a screen for thepresence or absence of malignancy or as a predictor of future cancer.

The use of antibodies of the present invention, in an ELISA assay iscontemplated. For example, anti-Killin antibodies are immobilized onto aselected surface, preferably a surface exhibiting a protein affinitysuch as the wells of a polystyrene microtiter plate. After washing toremove incompletely adsorbed material, it is desirable to bind or coatthe assay plate wells with a non-specific protein that is known to beantigenically neutral with regard to the test antisera such as bovineserum albumin (BSA), casein or solutions of powdered milk. This allowsfor blocking of non-specific adsorption sites on the immobilizingsurface and thus reduces the background caused by non-specific bindingof antigen onto the surface.

After binding of antibody to the well, coating with a non-reactivematerial to reduce background, and washing to remove unbound material,the immobilizing surface is contacted with the sample to be tested in amanner conducive to immune complex (antigen/antibody) formation.

Following formation of specific immunocomplexes between the test sampleand the bound antibody, and subsequent washing, the occurrence and evenamount of immunocomplex formation may be determined by subjecting sameto a second antibody having specificity for Killin that differs thefirst antibody. Appropriate conditions preferably include diluting thesample with diluents such as BSA, bovine gamma globulin (BGG) andphosphate buffered saline (PBS)/Tween®. These added agents also tend toassist in the reduction of nonspecific background. The layered antiserais then allowed to incubate for from about 2 to about 4 hr, attemperatures preferably on the order of about 25° to about 27° C.Following incubation, the antisera-contacted surface is washed so as toremove non-immunocomplexed material. A preferred washing procedureincludes washing with a solution such as PBS/Tween®, or borate buffer.

To provide a detecting means, the second antibody will preferably havean associated enzyme that will generate a color development uponincubating with an appropriate chromogenic substrate. Thus, for example,one will desire to contact and incubate the second antibody-boundsurface with a urease or peroxidase-conjugated anti-human IgG for aperiod of time and under conditions which favor the development ofimmunocomplex formation (e.g., incubation for 2 hr at room temperaturein a PBS-containing solution such as PBS/Tween®).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS)and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation isthen achieved by measuring the degree of color generation, e.g., using avisible spectrum spectrophotometer.

The preceding format may be altered by first binding the sample to theassay plate. Then, primary antibody is incubated with the assay plate,followed by detecting of bound primary antibody using a labeled secondantibody with specificity for the primary antibody.

The antibody compositions of the present invention will find great usein immunoblot or Western blot analysis. The antibodies may be used ashigh-affinity primary reagents for the identification of proteinsimmobilized onto a solid support matrix, such as nitrocellulose, nylonor combinations thereof. In conjunction with immunoprecipitation,followed by gel electrophoresis, these may be used as a single stepreagent for use in detecting antigens against which secondary reagentsused in the detection of the antigen cause an adverse background.Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies against the toxin moiety areconsidered to be of particular use in this regard.

V. Methods of Therapy

The present invention also involves, in another embodiment, thetreatment of cancer. The types of cancer that may be treated, accordingto the present invention, is limited only by the involvement of Killin.By involvement, it is not even a requirement that Killin be mutated orabnormal—the overexpression of this tumor suppressor may actuallyovercome other lesions within the cell. Thus, it is contemplated that awide variety of tumors may be treated using Killin therapy, includingcancers of the brain, lung, liver, spleen, kidney, lymph node, pancreas,small intestine, blood cells, colon, stomach, breast, endometrium,prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow,blood or other tissue.

In many contexts, it is not necessary that the tumor cell be killed orinduced to undergo normal cell death or “apoptosis.” Rather, toaccomplish a meaningful treatment, all that is required is that thetumor growth be slowed to some degree. It may be that the tumor growthis completely blocked, however, or that some tumor regression isachieved. Clinical terminology such as “remission” and “reduction oftumor” burden also are contemplated given their normal usage.

A. Genetic Based Therapies

One of the therapeutic embodiments contemplated by the present inventorsis the intervention, at the molecular level, in the events involved intumorigenesis. Specifically, the present inventors intend to provide, toa cancer cell, an expression construct capable of providing Killin tothat cell. Because the sequence homology between the human, mouse anddog genes, any of these nucleic acids could be used in human therapy, ascould any of the gene sequence variants discussed above which wouldencode the same, or a biologically equivalent polypeptide. The lengthydiscussion of expression vectors and the genetic elements employedtherein is incorporated into this section by reference. Particularlypreferred expression vectors are viral vectors such as adenovirus,adeno-associated virus, herpesvirus, vaccinia virus and retrovirus. Alsopreferred is liposomally-encapsulated expression vector.

Those of skill in the art are well aware of how to apply gene deliveryto in vivo and ex vivo situations. For viral vectors, one generally willprepare a viral vector stock. Depending on the kind of virus and thetiter attainable, one will deliver 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, 1×10¹¹ or 1×10¹² infectious particles to the patient.Similar figures may be extrapolated for liposomal or other non-viralformulations by comparing relative uptake efficiencies. Formulation as apharmaceutically acceptable composition is discussed below.

Various routes are contemplated for various tumor types. The sectionbelow on routes contains an extensive list of possible routes. Forpractically any tumor, systemic delivery is contemplated. This willprove especially important for attacking microscopic or metastaticcancer. Where discrete tumor mass may be identified, a variety ofdirect, local and regional approaches may be taken. For example, thetumor may be directly injected with the expression vector. A tumor bedmay be treated prior to, during or after resection. Following resection,one generally will deliver the vector by a catheter left in placefollowing surgery. One may utilize the tumor vasculature to introducethe vector into the tumor by injecting a supporting vein or artery. Amore distal blood supply route also may be utilized.

In a different embodiment, ex vivo gene therapy is contemplated. Thisapproach is particularly suited, although not limited, to treatment ofbone marrow associated cancers. In an ex vivo embodiment, cells from thepatient are removed and maintained outside the body for at least someperiod of time. During this period, a therapy is delivered, after whichthe cells are reintroduced into the patient; hopefully, any tumor cellsin the sample have been killed.

Autologous bone marrow transplant (ABMT) is an example of ex vivo genetherapy. Basically, the notion behind ABMT is that the patient willserve as his or her own bone marrow donor. Thus, a normally lethal doseof irradiation or chemotherapeutic may be delivered to the patient tokill tumor cells, and the bone marrow repopulated with the patients owncells that have been maintained (and perhaps expanded) ex vivo. Because,bone marrow often is contaminated with tumor cells, it is desirable topurge the bone marrow of these cells. Use of gene therapy to accomplishthis goal is yet another way Killin may be utilized according to thepresent invention.

B. Protein Therapy

Another therapy approach is the provision, to a subject, of Killinpolypeptide, active fragments, synthetic peptides, mimetics or otheranalogs thereof. The protein may be produced by recombinant expressionmeans or, if small enough, generated by an automated peptidesynthesizer. Formulations would be selected based on the route ofadministration and purpose including, but not limited to, liposomalformulations and classic pharmaceutical preparations.

C. Combined Therapy with Immunotherapy, Traditional Chemo- orRadiotherapy

Tumor cell resistance to DNA damaging agents represents a major problemin clinical oncology. One goal of current cancer research is to findways to improve the efficacy of chemo- and radiotherapy. One way is bycombining such traditional therapies with gene therapy. For example, theherpes simplex-thymidine kinase (HS-tk) gene, when delivered to braintumors by a retroviral vector system, successfully inducedsusceptibility to the antiviral agent ganciclovir (Culver et al., 1992).In the context of the present invention, it is contemplated that Killinreplacement therapy could be used similarly in conjunction with chemo-or radiotherapeutic intervention. It also may prove effective to combineKillin gene therapy with immunotherapy, as described above.

To kill cells, inhibit cell growth, inhibit metastasis, inhibitangiogenesis or otherwise reverse or reduce the malignant phenotype oftumor cells, using the methods and compositions of the presentinvention, one would generally contact a “target” cell with a Killinexpression construct and at least one other agent. These compositionswould be provided in a combined amount effective to kill or inhibitproliferation of the cell. This process may involve contacting the cellswith the expression construct and the agent(s) or factor(s) at the sametime. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the agent.

Alternatively, the gene therapy treatment may precede or follow theother agent treatment by intervals ranging from minutes to weeks. Inembodiments where the other agent and expression construct are appliedseparately to the cell, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the agent and expression construct would still be able to exert anadvantageously combined effect on the cell. In such instances, it iscontemplated that one would contact the cell with both modalities withinabout 12-24 hours of each other and, more preferably, within about 6-12hours of each other, with a delay time of only about 12 hours being mostpreferred. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several days (2, 3,4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse betweenthe respective administrations.

It also is conceivable that more than one administration of eitherKillin or the other agent will be desired. Various combinations may beemployed, where Killin is “A” and the other agent is “B”, as exemplifiedbelow:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/BOther combinations are contemplated. Again, to achieve cell killing,both agents are delivered to a cell in a combined amount effective tokill the cell.

Agents or factors suitable for use in a combined therapy are anychemical compound or treatment method that induces DNA damage whenapplied to a cell. Such agents and factors include radiation and wavesthat induce DNA damage such as, γ-irradiation, X-rays, UV-irradiation,microwaves, electronic emissions, and the like. A variety of chemicalcompounds, also described as “chemotherapeutic agents,” function toinduce DNA damage, all of which are intended to be of use in thecombined treatment methods disclosed herein. Chemotherapeutic agentscontemplated to be of use, include, e.g., adriamycin, 5-fluorouracil(5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C,cisplatin (CDDP) and even hydrogen peroxide. The invention alsoencompasses the use of a combination of one or more DNA damaging agents,whether radiation-based or actual compounds, such as the use of X-rayswith cisplatin or the use of cisplatin with etoposide. In certainembodiments, the use of cisplatin in combination with a Killinexpression construct is particularly preferred as this compound.

In treating cancer according to the invention, one would contact thetumor cells with an agent in addition to the expression construct. Thismay be achieved by irradiating the localized tumor site with radiationsuch as X-rays, UV-light, γ-rays or even microwaves. Alternatively, thetumor cells may be contacted with the agent by administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition comprising a compound such as, adriamycin, 5-fluorouracil,etoposide, camptothecin, actinomycin-D, mitomycin C, or more preferably,cisplatin. The agent may be prepared and used as a combined therapeuticcomposition, or kit, by combining it with a Killin expression construct,as described above.

Agents that directly cross-link nucleic acids, specifically DNA, areenvisaged to facilitate DNA damage leading to a synergistic,antineoplastic combination with Killin. Agents such as cisplatin, andother DNA alkylating agents may be used. Cisplatin has been widely usedto treat cancer, with efficacious doses used in clinical applications of20 mg/m² for 5 days every three weeks for a total of three courses.Cisplatin is not absorbed orally and must therefore be delivered viainjection intravenously, subcutaneously, intratumorally orintraperitoneally.

Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage. As such a number ofnucleic acid precursors have been developed. Particularly useful areagents that have undergone extensive testing and are readily available.As such, agents such as 5-fluorouracil (5-FU), are preferentially usedby neoplastic tissue, making this agent particularly useful fortargeting to neoplastic cells. Although quite toxic, 5-FU, is applicablein a wide range of carriers, including topical, however intravenousadministration with doses ranging from 3 to 15 mg/kg/day being commonlyused.

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageDNA, on the precursors of DNA, the replication and repair of DNA, andthe assembly and maintenance of chromosomes. Dosage ranges for X-raysrange from daily doses of 50 to 200 roentgens for prolonged periods oftime (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosageranges for radioisotopes vary widely, and depend on the half-life of theisotope, the strength and type of radiation emitted, and the uptake bythe neoplastic cells.

The skilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

The inventors propose that the local or regional delivery of Killinexpression constructs to patients with cancer will be a very efficientmethod for treating the clinical disease. Similarly, the chemo- orradiotherapy may be directed to a particular, affected region of thesubjects body. Alternatively, systemic delivery of expression constructand/or the agent may be appropriate in certain circumstances, forexample, where extensive metastasis has occurred.

In addition to combining Killin therapies with chemo- andradiotherapies, it also is contemplated that combination with other genetherapies will be advantageous. For example, targeting of Killin and p53mutations at the same time may produce an improved anti-cancertreatment. Any other tumor-related gene conceivably can be targeted inthis manner, for example, p21, Rb, APC, DCC, NF-1, NF-2, BCRA2, p16,FHIT, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, ras, myc, neu, raf,erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl.

It also should be pointed out that any of the foregoing therapies mayprove useful by themselves in treating a Killin. In this regard,reference to chemotherapeutics and non-Killin gene therapy incombination should also be read as a contemplation that these approachesmay be employed separately.

E. Formulations and Routes for Administration to Patients

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions—expression vectors, virus stocks,proteins, antibodies and drugs—in a form appropriate for the intendedapplication. Generally, this will entail preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well know inthe art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal or intravenous injection. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions,described supra. Of particular interest is direct intratumoraladministration, perfusion of a tumor, or administration local orregional to a tumor, for example, in the local or regional vasculatureor lymphatic system, or in a resected tumor bed.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

For oral administration the polypeptides of the present invention may beincorporated with excipients and used in the form of non-ingestiblemouthwashes and dentifrices. A mouthwash may be prepared incorporatingthe active ingredient in the required amount in an appropriate solvent,such as a sodium borate solution (Dobell's Solution). Alternatively, theactive ingredient may be incorporated into an antiseptic wash containingsodium borate, glycerin and potassium bicarbonate. The active ingredientmay also be dispersed in dentifrices, including: gels, pastes, powdersand slurries. The active ingredient may be added in a therapeuticallyeffective amount to a paste dentifrice that may include water, binders,abrasives, flavoring agents, foaming agents, and humectants.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

VI. Kits

According to the present invention, there are provided kits fordetecting killin mutations and Killin expression. The kit of the presentinvention can be prepared by known materials and techniques which areconventionally used in the art. Generally, kits comprises separate vialsor containers for the various reagents, such as probes, primers,enzymes, antibodies, etc. The reagents are also generally prepared in aform suitable for preservation by dissolving it in a suitable solvent.Examples of a suitable solvent include water, ethanol, various buffersolutions, and the like. The various vials or containers are often heldin blow-molded or injection-molded plastics.

VII. Transgenics

In one embodiment of the invention, transgenic animals are producedwhich contain a functional transgene encoding a functional Killinpolypeptide or variants thereof. Transgenic animals expressing Killintransgenes, recombinant cell lines derived from such animals andtransgenic embryos may be useful in methods for screening for andidentifying agents that induce or repress function of Killin. Transgenicanimals of the present invention also can be used as models for studyingindications such as cancers. The promoter controlling the transgene maybe one that is capable of tissue specific or inducible expression.Within a particularly preferred embodiment, transgenic mice aregenerated which overexpress Killin or express a mutant form of thepolypeptide.

In one embodiment of the invention, a Killin transgene is introducedinto a non-human host to produce a transgenic animal expressing a humanor murine Killin gene. The transgenic animal is produced by theintegration of the transgene into the genome in a manner that permitsthe expression of the transgene. Methods for producing transgenicanimals are generally described by Wagner and Hoppe (U.S. Pat. No.4,873,191; which is incorporated herein by reference), Brinster et al.(1985; which is incorporated herein by reference in its entirety) and in“Manipulating the Mouse Embryo; A Laboratory Manual” (1994); which isincorporated herein by reference in its entirety).

It may be desirable to replace the endogenous Killin by homologousrecombination between the transgene and the endogenous gene so as tomeasure the effects of only the transgene's expression. Typically, aKillin gene flanked by genomic sequences is transferred bymicroinjection into a fertilized egg. The microinjected eggs areimplanted into a host female, and the progeny are screened for theexpression of the transgene. Transgenic animals may be produced from thefertilized eggs from a number of animals including, but not limited toreptiles, amphibians, birds, mammals, and fish. Alternatively, theendogenous gene may be eliminated by deletion as in the preparation of“knock-out” animals, optionally followed by insertion of the Killintransgene. the absence of one or both alleles of a Killin gene in“knock-out” mice permits the study of the effects that a reduction in orloss of Killin protein has on a cell in vivo. Knock-out mice alsoprovide a model for the development of Killin-related cancers.

As noted above, transgenic animals and cell lines derived from suchanimals may find use in certain testing experiments. In this regard,transgenic animals and cell lines capable of expressing wild-type ormutant Killin may be exposed to test substances. These test substancescan be screened for the ability to enhance wild-type Killin expressionand or function or impair the expression or function of mutant Killin.

VIII. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Materials & Methods

Cell culture. The p53-inducible H1299 lung cancer cell line p53-3 was agift from XB Chen et al. (1996). Human colorectal adenocarcinoma celllines DLD-1 was from ATTC. DLD-1(tetR) (with a tetracycline-regulatedtransactivator) and DLD1 with an inducible wild-type p53 expression werekindly provided by Yu et al. (1999). Cos-1 cells were gift of J.Flanagan (Harvard Medical School). Cell culture conditions and theinductions of wild-type p53, GFP, and GFP-Killin expression in bothH1299 and DLD-1 cells were carried out essentially as describedpreviously (Yu et al., 1999; Chen et al., 1996; Stein et al., 2004).Proliferation assays were performed by seeding 5,000 cells in each wellof six-well tissue culture dishes in duplicate for each data point andincubated at 37° C. for 5 days, during which cells were trypsinized andcounted using the Coulter cell counter (Beckman Coulter).

Cell transfection, RNA isolation, FDD, Northern blot analysis, Real timeRT-PCR, and cDNA library screening. Cell transfection, FDD screening andNorthern Blot analysis were carried out essentially as describedpreviously (Stein et al., 2004). Quantitative real-time RT-PCR assaysfor endogenous killin mRNA expression were conducted with killinspecific primers, 5′-TACACAAGCACCCACATC-3′ and 5′-TACACAAGCACCCACATC-3′.The predicted killin-specific RT-PCR product is 151 by in size. Forsample normalization, a constitutively expressed house-keeping geneglyceraldehyde-3-phosphate dehydrogenase (GAPDH), was measured usingprimers, 5′-CATGAGAAGTATGACAACAGCCT-3′ and 5′-AGTCCTTCCACGATACCAAAGT-3′,which specifically amplify GAPDH mRNA. After DNase I treatment with aMessageClean kit (GenHunter) to remove any chromosomal DNA, total RNAfrom each sample was reverse-transcribed using an oligo dT₂₀ primer(GenHunter) and Superscriptase (Invitrogene). All real-time PCRreactions were performed on an iCycler iQ (Bio-Rad), using iQ™ SYBRGreen Supermix (Bio-Rad) as instructed by the manufacturer. Theamplification protocol consisted of 3 min denaturation at 95° C.followed by 45 cycles of amplification (95° C. for 10 s, 63° C. for 45s). Each sample was run in triplicate in separate wells to permitquantification of the killin and GAPDH mRNA expression. All data werecalculated by the comparative Ct method to determine relative geneexpression level. A 4.1 kb full-length killin cDNA was isolated from ahuman kidney cDNA library (Stratagene) using the killin FDD cDNA probeand completely sequenced.

RNA Interference. RNAi sequence targeting killin,5′-GGATACACGGGCCACAGTC-3′ (positions 153 to 171) was selected followingthe instruction from oligoengine.com. Primers used were: forward primer(64 mer) 5′-GATCCCCGGATACACGGGCCACAGTCTTCAAGAGAGACTGTGGCCCGTGTATCCTTTTTGGAAA-3′ and reverse primer (64mer)′-AGCTTTTCCAAAAAGGATACACGGGCCACAGTCTCTCTTGAAGACTGTGGC CCGTGTATCCGGG-3′. The annealed RNAi template was cloned into BglII andHindIII sites of pSUPER27 (gift from R. Agami, The Netherlands CancerInstitute, Amsterdam, Netherlands). The RNAi construct was confirmed bysequencing.

Dual Luciferase Reporter Assay. The p53 binding site located withinhuman Killin promoter was amplified by PCR from the genomic DNA of humanHEK293T cells using the following forward and reverse primers based onthe human genome database:

5′-GGTACCTCTGGGTGCGAGCGCAGAG-3′ 5′-AGATCTCGTTATCCTCGCCTCGCGTTG-3′

The 140 by PCR product was cloned first into PCR-TRAP cloning vector andthen shuttled into the KpnI and BglII sites of the pGL3-basic vector(Promega). H1299 cells (p53 null) were cultured overnight in 12-wellplate and subsequently co-transfected with pGL3 or pGL3-PKillin reporterplasmid with either pCEP4-p53 (p53) (Stein et al., 2004) expressingwild-type p53, or pRc-p53 expressing a loss of function mutant p53(p53-mut) (Taniura et al., 1999). Following 24 hrs of transfection,cells were lysed and the luciferase activity was determined using thedual luciferase reporter assay system (Promega) and Monolight™ 3010luminometer (BD PharMingen). All experiments were performed induplicate. The pRL-CMV plasmid containing the Renilla luciferasereporter gene (Promega) was used to normalize the transfectionefficiency.

Inducible GFP-Killin expression. The entire coding region of Killin (aa2-178) was amplified by PCR using primers:

B1 - 5′-CGCGGATCCGATCGCCCGGGGCCAGGCTCC-3′ B2 -5′-CGCGGATCCTCAGTCCTTTGGCTTGCTCTT-3′

After cloning into PCR-TRAP (GenHUnter), the insert was subcloned intothe BamHI site of pEGFP-C1 (Clontech) to allow in-frame fusion of Killinto EGFP. The GFP-Killin fusion was then shuttled into pTRE2 vector(Clonetech) as a NheI-HindIII fragment to allow tetracycline regulatedexpression after stably transfected into DLD-1 (TetR) cell line. GFPalone was also subcloned into pTRE2 from pEGFP-C 1 as a NheI-DraIfragment. All fusion constructs were verified by DNA sequencing.

FACS analysis, immunoblotting and fluorescent microscopy. FACS analysisand immunoblotting including sources of antibodies were essentially asdescribed previously (Stein et al., 2004). Fluorescent microscopy of GFPand GFP-killin expression in vivo was carried out using a Zeiss 200Minverted fluorescence microscope (Carl Zeiss Microimage, Germany) withtemperature- and CO₂-controlled chamber. Captured images were analyzedusing the Openlab software (Improvision, Lexington, Mass.). For confocalmicroscopy, cells were grown on cover slips, transfected with GFP-PCNAor GFP-Killin, fixed and stained with DAPI for DNA colocalization,mounted using the ProLong antifade (Molecular Probes) and examined witha Zeiss Axioplan 2 microscope using the 63× oil immersion objective.Digital images were captured with an ORCA-ER camera and selected imageswere processed by deconvolution microscopy using the OpenLab software.

DNA binding assays. In vitro transcribed and translated Killin clonedinto pcDNA3.1 (Invitrogen) (K), or vector alone (V) were labeled with³⁵S using Translabel (ICN) and a TnT kit (Promega). The labeled Killinand vector control were incubated with either single-stranded (ss) ordouble-stranded (ds) DNA cellulose (Sigma). After washing with PBS,bound proteins were resolved on a 15% SDS-PAGE gel, dried onto a 3Mpaper before autoradiography. For peptide binding, 42 aa of Killin/N8-50peptide (MW: 5007 dalton) was synthesized and verified with mass spec bySigma-Genosys at a purity of at least 70%. The peptide was dissolved inPBS as 1 mg/mL stock solution before use. For in vitro DNA bindingassay, 3 primers (32-35 bases in length) with arbitrary sequences weredesigned:

LL: 5′-TTTGCACGTCGGATCCGACCCAGACTACGGAGGCC-3′ RLM:5′-GGCCTCCGTAGTCTGGGTCGGATCCGACGTGC-3′ RL:5′-CCGGAGGCATCAGACGGTCGGATCCGACGTGC-3′)

After annealing, probes for artificial replication fork (LL and RL) ordouble-stranded DNA template (LL and RLM) were end-labeled withα-³²P-dATP (Perkin-Elmer) using Klenow (New England Biolab).Single-stranded oligonucleotide (LL) was end-labeled with γ-³²P-dATP(Perkin-Elmer) using T4 polynucleotide kinase (New England Biolab).After purifying with Sephadex-G50 spin columns (Roche), the labeledprobes (200,000 CPM each) were mixed with an increasing amount ofKillin/N8-50 peptide in the presence of 20 mM Tris-Cl (pH 8.4), 25 mMKCl, 1.5 mM MgCl₂ and 100 μg/mL of BSA. The reaction mixtures wereincubated at RT for 30 min and separated on a 6% polyacrylamide gel with1× TBE buffer. After drying the gel onto a Whatman No. 1 filter paper,the DNA-protein complexes were visualized by autoradiography. Forbinding kinetics, bands from DNA-peptide complex were excised and theradioactivity was determined by scintillation counting. To determine thestability of the DNA-Killin peptide complex, 0.5 μg of eitherdouble-stranded (RF form) or single-stranded (viral form) of PhiX174bacterial phage DNA (New England Biolab) were incubated at roomtemperature for 30 min with 1 ug of Killin/N8-50 peptide in a 20 μLreaction. The samples were then resolved on a 0.8% TAE agarose gel andstained with ethidium bromide.

In vitro DNA synthesis assay. pUC18 plasmid from exponentially growing(log phase) bacteria was purified and used as a template for in vitroDNA synthesis assay using a HotPrime DNA labeling kit (GenHunter). Foreach in vitro DNA synthesis assay, 0.8 μg of theta form (incompletelyreplicated) pUC18 was mixed with an increasing amount of Killin/N8-50peptide in the presence of 2.5 μCi of α-³²P-dATP and 1× labeling buffercontaining 200 μM of dNTP (-dATP). After 1 hour, 5 units of Klenow (NEB)were added to each reaction to initiate DNA synthesis reaction at 37° C.After 30 min, the reaction was stopped with 6 μl of 100 mM EDTA. MiniQuick DNA Spin columns (Roche Applied Science) were utilized to removeunincorporated nucleotides, and rate of DNA synthesis was determined byscintillation counting of purified high molecular weight DNA.

Random Mutagenesis, deletion analysis, and genetic screen of killin.Mutagenesis experiments were performed using EMS (ethylmethanesulfonate)(Sigma) as described previously with minor modification. Exponentiallygrowing XL-1 blue with pQE32-Killin was harvested by centrifugation,washed, and resuspended in minimal A buffer (10.5 g of K₂HPO₄, 4.5 g ofKH₂PO4, 1 g of [NH₄]₂SO4, and 0.5 g of sodium citrate:2H₂O in 1 liter).EMS was added to the cells at a concentration of 140 μM for 1 hourduring continuous shaking at 37° C. After washing with minimal A buffer,cells was diluted 1:10 in LB medium and grown for 6 hrs at 37° C. beforemutagenized plasmids were purified and transformed into either GH1(without repression, GenHunter) or XL-1 blue (with repression,Stratagene) competent cells. Ampicillin resistant colonies were scoredafter an overnight incubation.

For deletion analysis, PCR was performed to amplify different regions ofKillin as specified. PCR primer sequences used for the deletions arelisted below:

L-1 (32mer): 5′-CGCGGATCCTGGATCGCCCGGGGCCAGGCTCC-3′ L-8 (26mer):5′-GGATCCTGGCGCGCCCCGGCCGGACC-3′ L-11 (26mer):5′-GGATCCTGGGCCGGACCGTGCACGTT-3′ L-124 (26mer):5′-GGATCCTCCCGAAGGAGCGCTGTCGG-3′ R-43 (28mer):5′-GGATCCTCATAGGTCTCCTCGCCCCGCC-3′ R-49 (29mer):5′-GGATCCTACCTCCTTTTGAACCCTCCTAG-3′ R-85 (27mer):5′-GGATCCTAGCCTCCGGAGCTATCACTG-3′ R-97 (27mer):5′-GGATCCTAGGCAAGAGCACCCCGAGCA-3′ R-178 (30mer):5′-CGCGGATCCTCAGTCCTTTGGCTTGCTCTT-3′The obtained PCR products were first cloned into PCR-TRAP cloning vectorand then subcloned as BglII fragments into the BamHI site of the pQE326×HIS-tag expression vector (Qiagen). The expression vectors weretransformed into XL-1 blue under transcription repression for thetransgene (-IPTG). All killin deletion plasmid constructs were verifiedby DNA sequencing before being transformed into GH1 competent cells(GenHunter), which does not have lacI^(q) (no repression fortranscription), to allow scoring for inhibition of bacteria colonyformation when selected with ampicillin. As a negative control, allvectors were simultaneously transformed into XL-1 blue cells, which gavenear confluent transformants under transcription repression for allconstructs. Similar deletion constructs were also made as GFP fusionprotein as described above and transiently transfected into the H1299cells to visualized their ability to cause cell apoptosis based onnuclear chromosome condensation.

In vitro SV40 DNA replication assay. The SV40 origin (ori) ofreplication-dependent in vitro DNA replication was carried outessentially as described previously (Li and Kelly, 1984) with minormodifications. Briefly, the source of SV40 large T antigen was fromHEK293T cells (GenHunter). The SV40 ori-containing plasmid vectorpAPtag-2 (GenHunter) and a negative control vector pUC18 that lacks SV40ori were used as templates for the in vitro DNA replication assays. 10μg of nuclear protein extracts and 250 ng of plasmid DNA template wereused for each assay. Nascent DNA synthesis in the absence and presenceof Killin/N8-50 peptide after ³²P-α-dATP labeling was analyzed on a 6%TBE PAGE gel, dried onto a 3 M paper (Whatman) and visualize byautoradiography.

Example 2 Results

High-throughput fluorescent differential display (FDD) screening for p53target genes. In an attempt to systematically identify p53 target genesthat are involved in S-phase checkpoint control, the inventors employedthe comprehensive FDD screening strategy that they pioneered (Liang andPardee, 1992; Cho et al., 2001; Liang 2002; Liang and Pardee, 2003; Yangand Liang, 2004). They chose two cell types in which p53 mutations hadbeen clearly linked to human cancer, the p53-null human lung carcinomacell line H1299 (Chen et al., 1996) and the DLD-1 colon cancer cell line(Yu et al., 1999). Both cell lines contained tetracycline-regulatedexpression of wild-type p53 tumor suppressor gene and underwentapoptosis within 24-48 hrs following tetracycline withdraw (Chen et al.,1996; Yu et al., 1999; Stein et al., 2004). One of the greatestadvantages of Differential Display (DD) over many other methods fordifferential gene expression studies is that it allows simultaneouscomparison of more than just two RNA samples, so better controls can bebuilt in to cut down the biological variables or “noise” that arep53-independent (Liang and Pardee, 2003). For example, to control forthe effects of elapse of time, washing the cells and media change toremove tetracycline in order to turn on p53 expression, the inventorsincluded, in parallel, a control set of plates containing the same cellsthat were washed and incubated with new media with tetracycline (no p53induction). RNA and protein samples were isolated and the induction ofp53 and subsequent cell apoptosis were confirmed (FIGS. 1A-D). AfterDNase I treatment to remove any residual chromosomal DNA, four total RNAsamples from 9 and 12 hr time points without and with the induction ofp53 were reverse transcribed and processed for comprehensive FDDanalysis. After screening through 192 combinations of DD primers(G-anchor in combinations with arbitrary 13 mers HAP(1-120), A-anchorwith HAP(1-24) and C-anchor with HAP(1-48), over a dozen candidate p53target genes were identified (FIG. 8 & Table 6). This represented about40% coverage of all the genes expressed in a cell based on a recenttheoretical model of DD (Yang and Liang, 2004). DNA sequence analysisrevealed that 4 of them, G20, G54, G63 and G116 corresponded to thewild-type p53 transgene itself (Table 6). Among these were also severalknown bona fide p53 target genes, including human homolog of mdm-2(found twice, A10 and G10) and p21 (A21), while the rest of thecandidate p53 target genes including G101 (killin) and NDRG1 were eithernovel genes or novel p53 targets (Stein et al., 2004) (Table 6). Thefindings of p53 induction as well as other major known p53 target genesseveral times by FDD demonstrated an excellent gene coverage andaccuracy of our FDD platform, since the method is non-biased and doesnot require any prior knowledge in gene sequences being detected (Yangand Liang, 2004). While the inventors have previously shown that NDRG1is necessary for p53-mediated apoptosis (Stein et al., 2004), here theinventors focus on the characterization of killin and show that it is anovel p53 target gene which functions directly in S-phase control andapoptosis.

TABLE 6 p53 target genes identified by comprehensive FDD ScreeningAnchor FDD band Gene identity used H-AP used Known Target G3 NDRG1 GH-AP-3 Yes G7 Novel G H-AP-3 No G10 HDM-2 G H-AP-10 Yes G14 Novel GH-AP-14 No G17 NDRG1 G H-AP-17 Yes G20 p53 G H-AP-20 Yes G29 Pir-121 GH-AP-29 Yes-No G40 Glutaminase G H-AP-40 No G54 p53 G H-AP-54 Yes G63p53 G H-AP-63 Yes G77 Novel G H-AP-77 No G101 (killin) Novel G H-AP-101No G116 p53 G H-AP-116 No A9 PP2C-gamma-like A H-AP-10 No A10 HDM-2 AH-AP-10 Yes A21 p21 A H-AP-21 Yes C25 Tis11d C H-AP-25 No C26 Novel CH-AP-26 No C29 Pir-121 C H-AP-29 Yes-no

Identification of killin as a novel p53 target gene. The identificationof killin as a p53 target gene by FDD and its confirmation by Northernblot analysis using the cDNA fragment recovered from FDD was shown inFIGS. 1A-B. The induction of the 4 kb killin mRNA following tetracyclinewithdraw was evident from 9 hrs, which was slightly behind the inductionof p53 as expected. To rule out the effect of tetracycline on killinexpression that was independent of p53, H1299 (p53 null) parental cellsgrown in the absence of tetracycline was included as a negative control,which confirmed that killin expression was indeed p53-dependent (FIG.1C). DNA sequence analysis of the 542 by killin cDNA recovered from FDDrevealed that killin is a novel gene, which is normally expressed at lowlevel detectable only in kidney and lung. Using the killin FDD cDNA as aprobe, a 4.1 kb full-length cDNA for the gene was isolated from a humankidney cDNA library. After complete sequencing, the cDNA was shown toencode a novel and small 20 kDa basic protein of 178 amino acids with anapparent pI of 11.3 (FIG. 1D). An in-frame stop codon was found within15 bases upstream of the predicted translation start site of Killin.Bioinformatic analysis of Killin indicated that the protein did notshare any homology to any known proteins from any species, except twoputative nuclear localization domains were noted (FIG. 1D).

Killin is localized near the pTEN tumor-suppressor gene locus whichcontains a divergent promotor driving p53-dependent transcription ofkillin. DNA sequencing and genomic database search revealed that killinis localized in close proximity to the pten tumor-suppressor gene onhuman chromosome 10 (FIG. 2A). In fact, the intergenic region separatingthe two genes (based on transcriptional start sites) is only 194 by inlength that contains a divergent promoter with a highly consensus p53binding site (FIG. 2A). Interestingly, pTEN was previously shown to bemodulated by p53 as well, although, unlike killin, the basal level ofPTEN expression appears to be constitutive (Stambolic et al., 2001). Inorder to determine if killin is a direct p53 target gene, a dualluciferase reporter assay was performed using the 140 by intergenicregion containing the conserved p53-binding site (FIG. 2B). The killinpromoter conferred about 70-fold increase in wild-type p53-dependentluciferase activity, whereas an expression vector encoding a DNA-bindingmutant p53 (R248W) failed to activate the promoter. Moreover, mutationswithin the conserved p53 binding site in the killin promoter greatdecreased the p53-dependent promoter strength (FIG. 2B). Taken together,these detailed promoter analysis confirms that killin is a directtranscriptional target of p53.

Killin is localized in the cell nucleus. To shed light on the biologicalfunction of Killin, the inventors first tried to determine itssubcellular localization. Given its alkaline pI and the existence of twoputative nuclear localization domains, the surmised that Killin might bea nuclear protein. To confirm this prediction, a GFP-Killin in-framefusion protein was constructed. After stable transfection into the DLD-1colon cancer cell line with a Tet repressor, the induction of either GFPalone or GFP-Killin within 16 hrs after removal of tetracycline wasvisualized under a fluorescence microscope. In contrast to GFP alone,which was expressed throughout the cells, GFP-Killin was exclusivelynuclear in localization as shown by DAPI co-staining of the nuclei (FIG.3A). To better visualize the sub-cellular distribution of Killin in thenucleus, which could provide clues to its biochemical functions, theinventors transiently transfected the GFP-Killin expression vector intoCos-1 cells, which were well attached and had large nuclei as depictedby DAPI staining (FIG. 3B). GFP-Killin was clearly localized in thenucleus of transfected cells and appeared to present as nuclear clustersor foci (FIG. 3B, upper left). Such focal distribution of GFP-Killinseemed to precede the nuclear condensation of apoptotic cells (FIG. 3B,upper right). Confocal fluorescent microscopy of Cos-1 cells expressingeither GFP-PCNA or GFP-Killin provided a higher resolution of phasespecific distribution of the corresponding proteins as nuclear foci(FIG. 3C). For GFP-PCNA, these foci are known to be clusters ofreplication forks along the chromatin where PCNA binds in S-phase nuclei(Leonhardt et al., 2000), whereas GFP-Killin foci exhibited a similarcontinuous cluster distribution pattern, except that the foci appearedmore diffusive. These data suggest that Killin appears to be associatedwith chromatin.

Killin is necessary for p53-mediated apoptosis. To determine if Killinis involved in any of the p53-mediated biological functions, theinventors employed RNAi technology to selectively knockdown the killinmRNA expression in H1299 cell line containing an inducible wild-type p53gene, which was used for the initial FDD screening. Compared to cellsstably transfected with the pSUPER RNAi vector control, cells stablytransfected with pSUPER-RNAi-Killin showed not only a significantlydecreased killin mRNA expression, but also marked blockade ofp53-mediated apoptosis manifested by dramatic inhibition of bothcaspase-3 activation and caspase-dependent PARP cleavage, as well as byFACS analysis of the cell cycle profiles (FIGS. 4A-B). Moreover,blocking killin expression had little effect on p53 induced p21expression (FIG. 4A), which led to mainly G1 arrest of the cells, asexpected (FIG. 4B).

Killin is sufficient in inducing cell Growth arrest at S-phase followedby massive apoptosis. To determine if Killin is sufficient in triggeringcell growth arrest and apoptosis, the inventors next analyzed the effectof inducible expression of GFP-Killin over time in DLD-1 colon cancercells. Based on measurements of cell proliferation, fluorescentmicroscopy and FACS analysis, GFP-Killin was shown to cause rapid cellgrowth arrest within 24 hrs after tetracycline removal, whereas GFPalone had little effect (FIG. 5A). Interestingly, unlike p53-mediatedgrowth arrest which occurs primarily at G1 via p21 (Chen et al., 1996),FACS analysis indicated that there was little decrease in S-phase DNAcontent nor increase in either G1 or G2 DNA content during the first 48hrs of cell growth arrest following the induction of Killin (FIG. 5B).This rather surprising finding suggests that Killin may function as aninhibitor of DNA replication and causes S-phase arrest. However, massiveapoptosis was observed by FACS analysis and fluorescence microscopyafter 2-3 days following tetracycline removal and the induction ofGFP-Killin (FIGS. 5B-C). This finding suggests that Killin inducedgrowth arrest is coupled to cell death, in contrast to GI arrestmediated by p21, which prevents cells from undergoing apoptosis.

Killin is a high affinity DNA binding protein. Based on Killin's nuclearlocalization, pattern of distribution and its potent effect on cellgrowth arrest at S-phase, the inventors hypothesized that Killin is aDNA binding protein. To further biochemically and functionallycharacterize Killin, four different experimental approaches were takento verify this prediction. First, the inventors tried to bacteriallyexpress and purify a 6×HIS-tagged Killin. It turned out that theinduction of the recombinant fusion protein by IPTG caused immediategrowth arrest of the bacteria hosts within 30 min when the expressedprotein could be barely detectable by Western blot analysis usingHIS-tag monoclonal antibody. In addition to extremely low-levelexpression before cells stopped growing, bacterially expressed Killinappeared to adopt a unique conformation (e.g., association with othermolecules such as DNA), which prevents it from being able to bind toNi-NTA column, making its purification in native form literallyimpossible. The extremely toxic effect of low level Killin expression inbacteria appeared to concur with our prediction for it being a generalDNA synthesis inhibitor, given the fact that bacteria have naked DNA. Toovercome the difficulty in expression and purifying Killin protein, thefull length Killin with predicted 20 kDa molecular weight was producedby in vitro transcription and translation, and shown to be able to bindto both single-stranded and double-stranded DNA templates (FIG. 6A). Incontrast, a higher molecular weight non-specific protein encoded by thevector alone or the free labels failed to bind to the DNA cellulosebeads, which served as negative controls for binding specificity (FIG.6A).

Genetic and biochemical mapping of the DNA binding domain of killin. Tofurther confirm and better define the functional domain of Killin forDNA binding, the inventors then turned to a genetic approach by takingadvantage of the toxicity of Killin expression in E. coli. Theyhypothesized that the toxicity of Killin in mammalian cells and bacteriaare functionally related and might have something to do with its abilityto bind DNA and inhibit DNA replication. Thus, they first truncated thecoding region of Killin into two parts at a unique Eco47III restrictionsite and showed that the N-terminal 123 amino acids were sufficient toretain toxicity to E. coli, whereas the plasmid expressing theC-terminal 124-178 amino acid residues was able to transform E. coliinto colonies in the absence of transcriptional repression (FIG. 6B). Tospeed up the genetic screen for the functional domain of Killin, theythen randomly mutagenized the plasmid encoding the full-lengthN-terminal HIS-tagged Killin using an alkylating agent,ethylmethanesulfonate (EMS). Compared to non-mutagenized vector, themutagenesis allowed colony formation following transformation into awild-type lac I host. DNA sequencing analysis revealed that the loss offunction mutations of killin fell into 5 groups, and they were eitherpremature nonsense mutations at codon 18, 24, 33 and 37 (FIG. 6B), orwith a deletion of a tandem-repeat sequence within the promoter regionof the pQE32 bacterial expression vector. The concentration of the lossof function mutations near the N-terminus of Killin suggested that itsfunctional domain is smaller than initially anticipated. Further refineddeletional mutagenesis was then conducted by PCR from both the N- andC-terminus of Killin and the results allowed the inventors tounambiguously pinpoint the minimal sequence from 8-49 amino acidresidues, which were essential for Killin's toxicity in bacteria (FIG.6B). It did not escape our notice that this region of Killin containedmultiple WXXR or KXXW motifs and is rich in basic amino acids (FIG. 6C).

To overcome the difficulty in high-level expression of Killin due to itstoxicity, the inventors chemically synthesized a peptide of 42 aminoacid residues in length corresponding to N8-50 of Killin (FIG. 6C, hereunder designated as Killin/N8-50). In vitro kinetic binding studiesusing ³²P-end labeled oligo nucleotide probes demonstrated thatKillin/N8-50 peptide was able to bind to double-stranded DNA and anartificial replication fork with an apparent Kd of 1 μM, whereas theaffinity to single-stranded DNA template appeared to be slightly higherwith an apparent Kd being 0.5 μM (FIG. 6D). This important findingprovides a biochemical basis for Killin function.

Killin forms a highly stable non-covalently linked complex with DNA andinhibits DNA synthesis in vitro. Killin could bind not only to oligonucleotide probes, but also much larger templates such as bacteria phageDNA and plasmids, which could be easily visualized by ethidium bromidestaining (FIG. 7A). Complexes formed between Killin/N8-50 peptide andeither the double-stranded or single-stranded bacteria phage PhiX174 DNAwere so stable that it could withstand 6M urea and 150 mM EDTA,suggesting that neither hydrogen bonds nor divalent cations wereinvolved in the interaction (FIG. 7A). However, SDS at a concentrationas low as 0.1% could completely disrupt the Killin-DNA complex,indicating that Killin/N8-50 was neither a nuclease, nor the Killin-DNAcomplex was covalently bound.

To determine if Killin/N8-50 peptide binding to DNA has any consequencesin DNA replication, the inventors employed the commonly used in vitroeukaryotic DNA replication assays originally described by Li and Kelly(1984). This assay uses soluble cell-free system derived from mammaliancell nuclear extract that is capable of replicating exogenous plasmidDNA molecules containing the simian virus 40 (SV40) origin ofreplication. Replication in the system is completely dependent upon theaddition of the SV40 large T antigen. Using this assays, the inventorsshowed that the Killin/N8-50 peptide could greatly inhibit DNAreplication (FIG. 7B). The requirement of higher concentration ofKillin/N8-50 peptide for the inhibition of DNA replication than thatseen in the in vitro DNA binding assays was most likely due to the highconcentration of chromosomal DNA present in the nuclear extracts used asa source of SV40 large T antigen. Such chromosomal DNA would conceivablycompete against plasmid template for Killin peptide binding, thuscompetitively inhibit the plasmid DNA replication. This prediction wasconsistent with results obtained by decreasing the amount nuclearextract used for the assay (data not shown).

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and/or methods and in the steps or in the sequence of stepsof the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

IX. References

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

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1-6. (canceled)
 7. A nucleic acid of about 15 to about 5000 base pairscomprising from about 15 contiguous base pairs of SEQ ID NO:2, or thecomplement thereof.
 8. The nucleic acid of claim 7, comprising fromabout 20 contiguous base pairs of SEQ ID NO:2, or the complementthereof.
 9. The nucleic acid of claim 7, comprising from about 30contiguous base pairs of SEQ ID NO:2, or the complement thereof.
 10. Thenucleic acid of claim 7, comprising from about 50 contiguous base pairsof SEQ ID NO:2, or the complement thereof.
 11. The nucleic acid of claim7, comprising about 100 contiguous base pairs of SEQ ID NO:2, or thecomplement thereof.
 12. The nucleic acid of claim 7, comprising about150 contiguous base pairs of SEQ ID NO:2.
 13. The nucleic acid of claim7, comprising about 250 contiguous base pairs of SEQ ID NO:2, or thecomplement thereof.
 14. The nucleic acid of claim 7, comprising about500 contiguous base pairs of SEQ ID NO:2, or the complement thereof. 15.The nucleic acid of claim 7, comprising about 1000 contiguous base pairsof SEQ ID NO:2, or the complement thereof.
 16. The nucleic acid of claim7, comprising about 2500 contiguous base pairs of SEQ ID NO:2, or thecomplement thereof.
 17. The nucleic acid of claim 7, comprising about4000 contiguous base pairs of SEQ ID NO:2, or the complement thereof.18. A peptide comprising about 10-50 contiguous amino acids of SEQ IDNO:1.
 19. The peptide of claim 18, comprising about 15 contiguous aminoacids of SEQ ID NO:1.
 20. The peptide of claim 18, comprising about 25contiguous amino acids of SEQ ID NO:1.
 21. The peptide of claim 18,comprising about 50 contiguous amino acids of SEQ ID NO:1.
 22. Thepeptide of claim 18, comprising residues 8-49 of of SEQ ID NO:1.
 23. Apolypeptide comprising the sequence of SEQ ID NO:1.
 24. An expressioncassette comprising a polynucleotide encoding a polypeptide having thesequence of SEQ ID NO:1 or a fragment thereof, wherein saidpolynucleotide is under the control of a promoter operable in eukaryoticcells. 25-33. (canceled)
 34. A method for suppressing growth of a cancercell comprising contacting said cells with an expression cassettecomprising a polynucleotide encoding a polypeptide having the sequenceof SEQ ID NO:1 or a fragment thereof, wherein said polynucleotide isunder the control of a promoter operable in eukaryotic cells.
 35. Themethod of claim 34, wherein said promoter is heterologous to thepolynucleotide sequence.
 36. The method of claim 35, wherein saidpromoter is selected from the group consisting of hsp68, SV40, CMV, MKC,GAL4_(UAS), HSV and β-actin.
 37. The method of claim 35, wherein saidpromoter is a tissue specific promoter.
 38. The method of claim 35,wherein said promoter is an inducible promoter.
 39. The method of claim35, wherein said expression cassette is contained in a viral vector. 40.The method of claim 35, wherein said viral vector is selected from thegroup consisting of a retroviral vector, an adenoviral vector, andadeno-associated viral vector, a vaccinia viral vector, and aherpesviral vector.
 41. The method of claim 34, wherein said expressioncassette further comprises a polyadenylation signal.
 42. The method ofclaim 34, wherein said expression cassette comprises a secondpolynucleotide encoding a second polypeptide.
 43. The method of claim42, wherein said second polynucleotide is under the control of a secondpromoter.
 44. A cell comprising an expression cassette comprising apolynucleotide encoding a polypeptide having the sequence of SEQ ID NO:1or a fragment thereof, wherein said polynucleotide is under the controlof a promoter operable in eukaryotic cells.
 45. A monoclonal antibodythat binds immunologically to a polypeptide having the sequence of SEQID NO:1, or an immunologic fragment thereof. 46-49. (canceled)
 50. Amethod of diagnosing a cancer comprising the steps of: (i) obtaining atissue sample from a subject; and (ii) assessing the expression orstructure of Killin in cells of said sample.
 51. The method of claim 50,wherein said cancer is selected from the group consisting of brain,lung, liver, spleen, kidney, lymph node, small intestine, pancreas,blood cells, colon, stomach, breast, endometrium, prostate, testicle,ovary, skin, head and neck, esophagus, bone marrow and blood cancer. 52.The method of claim 50, wherein step (ii) comprises assessing Killinexpression.
 53. The method of claim 50, wherein step (ii) comprisesassessing Killin structure.
 54. The method of claim 50, wherein saidcancer is a colon cancer or lung cancer.
 55. The method of claim 50,wherein said sample is a tissue or fluid sample.
 56. The method of claim50, wherein said assessing comprises assaying for a Killin-encodingnucleic acid from said sample.
 57. The method of claim 56, furthercomprising subjecting said sample to conditions suitable to amplify saidnucleic acid.
 58. The method of claim 50, wherein said assessingcomprises contacting said sample with an antibody that bindsimmunologically to a Killin polypeptide.
 59. The method of claim 58,further comprising subjecting proteins of said sample to ELISA.
 60. Themethod of claim 50, wherein assessing involves evaluating the level ofKillin expression.
 61. The method of claim 50, further comprising thestep of comparing the expression of Killin with the expression of Killinin non-cancer samples.
 62. The method of claim 50, wherein assessinginvolves evaluating the structure of the Killin gene or transcript. 63.The method of claim 62, wherein said evaluating comprises an assayselected from the group consisting of sequencing, wild-typeoligonucleotide hybridization, mutant oligonucleotide hybridization,SSCP, PCR and RNase protection.
 64. The method of claim 63, wherein asaid evaluating is wild-type or mutant oligonucleotide hybridization andsaid oligonucleotide is configured in an array on a chip or wafer.
 65. Amethod for altering the phenotype of a tumor cell comprising the step ofadministering to a cell a tumor suppressor designated Killin or afragment thereof under conditions permitting the uptake of said tumorsuppressor by said tumor cell.
 66. The method of claim 65, wherein saidtumor cell is derived from a tissue selected from the group consistingof brain, lung, liver, spleen, kidney, lymph node, small intestine,blood cells, pancreas, colon, stomach, breast, endometrium, prostate,testicle, ovary, skin, head and neck, esophagus, bone marrow and bloodtissue. 67-68. (canceled)
 69. A method for altering the phenotype of atumor cell comprising the step of contacting the cell with a nucleicacid (i) encoding a tumor suppressor designated Killin or a fragmentthereof and (ii) a promoter active in said tumor cell, wherein saidpromoter is operably linked to the region encoding said tumorsuppressor, under conditions permitting the uptake of said nucleic acidby said tumor cell.
 70. The method of claim 69, wherein said tumor cellis derived from a tissue selected from the group consisting of brain,lung, liver, spleen, kidney, lymph node, small intestine, blood cells,pancreas, colon, stomach, breast, endometrium, prostate, testicle,ovary, skin, head and neck, esophagus, bone marrow and blood tissue. 71.The method of claim 70, wherein the a phenotype is selected from thegroup consisting of apoptosis, angiogenesis, proliferation, migration,contact inhibition, soft agar growth or cell cycling.
 72. The method ofclaim 70, wherein said nucleic acid is encapsulated in a liposome. 73.The method of claim 70, wherein said nucleic acid is a viral vectorselected from the group consisting of retrovirus, adenovirus,adeno-associated virus, vaccinia virus and herpesvirus.
 74. The methodof claim 73, wherein said nucleic acid is encapsulated in a viralparticle.
 75. A method for treating subject with cancer comprising thestep of administering to said subject a tumor suppressor designatedKillin or a fragment thereof.
 76. The method of claim 75, wherein saidtumor cell is derived from a tissue selected from the group consistingof brain, lung, liver, spleen, kidney, lymph node, small intestine,blood cells, pancreas, colon, stomach, breast, endometrium, prostate,testicle, ovary, skin, head and neck, esophagus, bone marrow and bloodtissue.
 77. (canceled)
 78. A method for treating a subject with cancercomprising the step of administering to said subject a nucleic acid (i)encoding a tumor suppressor designated Killin or a fragment thereof and(ii) a promoter active in eukaryotic cells, wherein said promoter isoperably linked to the region encoding said tumor suppressor.
 79. Themethod of claim 78, wherein said tumor cell is derived from a tissueselected from the group consisting of brain, lung, liver, spleen,kidney, lymph node, small intestine, blood cells, pancreas, colon,stomach, breast, endometrium, prostate, testicle, ovary, skin, head andneck, esophagus, bone marrow and blood tissue.
 80. (canceled)
 81. Anon-human transgenic eukaryote lacking a functional Killin gene. 82.(canceled)
 83. A non-human transgenic eukaryote that overexpressesKillin as compared to a similar non-transgenic eukaryote.
 84. (canceled)85. A method of screening a candidate substance for anti-tumor activitycomprising the steps of: (i) providing a cell lacking functional Killinpolypeptide; (ii) contacting said cell with said candidate substance;and (iii) determining the effect of said candidate substance on saidcell. 86-94. (canceled)
 95. An isolated and purified nucleic acid thathybridizes, under high stringency conditions, to a DNA segmentcomprising SEQ ID NO:2.
 96. The method of claim 75, further comprisingtreating said subject with a second anti-cancer therapy. 97-98.(canceled)
 99. The method of claim 78, further comprising treating saidsubject with a second anti-cancer therapy. 100-101. (canceled)
 102. Amethod of screening a candidate substance for anti-tumor activitycomprising the steps of: (i) providing a cell expression a functionalKillin peptide or polypeptide; (ii) contacting said cell with saidcandidate substance; and (iii) determining Killin DNA binding or nuclearlocalization, wherein an increase in Killin DNA binding or nuclearlocalization, as compared to a similar cell not treated with saidcandidate substance, indicates that said candidate substance hasanti-tumor activity.
 103. A nucleic acid segment comprising SEQ ID NO:3.104. A method of screening for an activator of Killin expressioncomprising: (i) providing a cell comprising a Killin promoter operablylinked to a nucleic acid segment encoding expressible marker; (ii)contacting said cell with a candidate substance; and (iii) assessing theexpression of said marker, wherein an increase in expression of saidmarker, as compared to expression in a cell not contacted with saidcandidate substance, identifies said candidate substance as an activatorof Killin expression. 105-107. (canceled)